Abstracts of papers presented at the
EMBO Conference Series: Protein Synthesis and
Translational Control
EMBL Advanced Training Centre, Heidelberg, Germany, 08 - 12 Sep 2013
Scientific Organisers:
Fátima Gebauer, Centre for Genomic Regulation (CRG) and UPF, Spain
Matthias Hentze, EMBL Heidelberg, Germany
Alan Hinnebusch, National Institutes of Health, USA
Marat Yusupov, IGBMC, France
Conference Organiser:
Tim Nürnberger, EMBL Heidelberg, Germany
Cover illustration and poster by
Petra Riedinger, EMBL Heidelberg, Germany
Layout and abstract book preparation by
Tim Nürnberger, EMBL Heidelberg, Germany
Printed by Mera Druck GmbH, Sandhausen, Germany
These abstracts should not be cited in bibliographies. Material contained herein should be treated as
personal communication and should be cited as such only with the consent of the author.
The European Molecular Biology Laboratory wishes to thank
the members of the EMBL Advanced Training Centre Corporate
Partnership Programme.
Their support makes it possible to host conferences and courses
on emerging topics and it enables us to continue to offer excellent
scientific quality of our events.
If your organisation would like to receive information about the EMBL Advanced
Training Centre Corporate Partnership Programme, please contact Antonio Costantino
at [email protected]
Thank you to our sponsors:
Event Sponsors
Zymo Research Europe GmbH, Germany
The Company of Biologists, UK
Institute for Research in Biomedicine, Spain
Media Partners
BioMed Central, UK
Cold Spring Harbor Laboratory, USA
Open Biology, A Royal Society open access journal, UK
Technology Networks Ltd, UK
Session Overview
Sunday 08 September 2013
16:00 - 18:00
Registration
ATC Reception
18:00 - 19:00
Dinner
ATC Canteen
19:00 - 19:15
Welcome
Klaus Tschira Auditorium
19:15 - 20:00
Keynote History Lecture: Nahum Sonenberg
Klaus Tschira Auditorium
20:00 - 20:45
Keynote Lecture: Nenad Ban
Klaus Tschira Auditorium
20:45 - 22:45
Welcome Reception & Meet the Speakers
ATC Foyer
Monday 09 September 2013
09:00 - 12:30
Session 1: Regulation of initiation
Chair: Martin Bushell
Klaus Tschira Auditorium
12:30 - 14:00
Lunch
ATC Foyer
14:00 - 17:30
Session 2: Elongation, Termination and Recycling
Chair: Tatyana Pestova
Klaus Tschira Auditorium
17:30 - 18:00
Refreshments
ATC Foyer
18:00 - 19:30
Dinner
ATC Canteen
19:30 - 21:30
Poster Session I
ATC Helices
Page 1
EMBO Conference Series: Protein Synthesis and Translational Control
21:30 - 23:00
Wine & Cheese and Meet the Speakers
ATC Foyer
Tuesday 10 September 2013
09:00 - 11:45
Session 3: Ribonucleoprotein Complexes
Chair: Jernej Ule
Klaus Tschira Auditorium
11:45 - 16:30
Free Time/ Sightseeing Downtown
16:30 - 19:15
Session 4: Mechanistic insights into initiation
Chair: Nicola Gray
Klaus Tschira Auditorium
19:15 - 20:30
Dinner
ATC Canteen
20:30 - 22:30
Poster Session II
ATC Helices
22:30 - 23:30
Wine & Cheese and Meet the Speakers
ATC Foyer
Page 2
Session Overview
Wednesday 11 September 2013
09:00 - 12:30
Session 5: The Ribosome
Chair: Roland Beckmann
Klaus Tschira Auditorium
12:30 - 14:00
Lunch
ATC Foyer
14:00 - 16:00
Poster Session III
ATC Helices
16:00 - 18:15
Session 6: RNA Processing and Turnover
Chair: Oliver Mühlemann
Klaus Tschira Auditorium
18:15 - 19:15
Pre-Dinner Drinks & Meet the Speakers
ATC Foyer
19:15 - 01:00
Conference Dinner & Party
ATC Canteen and Foyer
Thursday 12 September 2013
09:15 - 12:00
Session 7: Non-coding RNA and Development
Chair: Antonio Giraldez
Klaus Tschira Auditorium
12:00 - 12:15
Closing Remarks
Klaus Tschira Auditorium
12:15 - 13:15
Packed Lunch & Departure
Page 3
EMBO Conference Series: Protein Synthesis and Translational Control
Page 4
Programme
Sunday 08 September 2013
16:00 - 18:00
Registration
ATC Reception
18:00 - 19:00
Dinner
ATC Canteen
19:00 - 19:15
Welcome
Klaus Tschira Auditorium
19:15 - 20:00
A historical perspective: translation initiation in eukaryotes
in health and disease
Nahum Sonenberg
McGill University, Canada
1
20:00 - 20:45
Structural studies of eukaryotic ribosomes and functional
insights
Nenad Ban
ETH Zurich, Switzerland
2
20:45 - 22:45
Welcome Reception & Meet the Speakers
ATC Foyer
Page 5
EMBO Conference Series: Protein Synthesis and Translational Control
Monday 09 September 2013
09:00 - 12:30
Session 1: Regulation of initiation
Chair: Martin Bushell
Klaus Tschira Auditorium
09:00 - 09:30
microRNA mechanism: Are we there yet?
Martin Bushell
MRC Toxicology Unit, United Kingdom
3
09:30 - 09:45
eIF2B displaces eIF5 from eIF2•GDP to promote efficient
nucleotide exchange and activation of eIF2
Martin Jennings
University of Manchester, United Kingdom
4
09:45 - 10:00
eIF4G and eIF1 cooperate to facilitate translation start
codon selection
Rivka Dikstein
The Weizmann Institute of Science, Israel
5
10:00 - 10:15
CLIP-seq analysis of the eIF4F complex reveals principles
of mRNP formation and regulation
David Weinberg
Whitehead Institute / MIT, United States of America
6
10:15 - 10:30
Positional proteomics and ribosome profiling to map the
translation initiation landscape
Petra Van Damme
University of Ghent, Belgium
7
10:30 - 11:00
Coffee Break
ATC Foyer
11:00 - 11:15
mTORC1-regulated translation of the C/EBPß-mRNA
determines health- and life-span in mice
Cornelis Calkhoven
Leibniz Institute for Age Research - Fritz Lipmann Institute,
Germany
Page 6
8
Programme
11:15 - 11:30
An upstream open reading frame in ERCC5 establishes
resistance to Cisplatin
Joanna Somers
MRC Toxicology Unit, United Kingdom
11:30 - 11:45
CGG repeat associated Non-AUG initiated translation
9
10
Peter Todd
University of Michigan, United States of America
11:45 - 12:00
Cellular mRNAs access second ORFs using a novel amino
acid sequence-dependent coupled translation
termination-reinitiation mechanism
Phillip Gould
University of Warwick, United Kingdom
11
12:00 - 12:15
TOR signaling regulates reinitiation of translation
Mikhail Shchepetilnikov
Université de Strasbourg, France
12
12:15 - 12:30
Functional profiling of IRES elements during cellular
hypoxia by translation of circular RNAs
Eric Mills
Johns Hopkins University School of Medicine, United States
of America
13
12:30 - 14:00
Lunch
ATC Foyer
14:00 - 17:30
Session 2: Elongation, Termination and Recycling
Chair: Tatyana Pestova
Klaus Tschira Auditorium
14:00 - 14:30
Mechanisms of eukaryotic reinitiation
Tatyana Pestova
SUNY Downstate Medical Center, United States of America
Page 7
14
EMBO Conference Series: Protein Synthesis and Translational Control
14:30 - 14:45
Accuracy of decoding on the ribosome is maintained by
insisting on Watson-Crick geometry
Natalia Demeshkina
IGBMC, France
15
14:45 - 15:00
GTP hydrolysis by EF-G synchronizes tRNA translocation
on small and large ribosomal subunits
Wolf Holtkamp
MPI for Biophysical Chemistry, Germany
16
15:00 - 15:15
Structure and structural dynamics of mammalian
ribosomal complexes during translation elongation
Christian Spahn
Charite - Universitätsmedizin Berlin, Germany
17
15:15 - 15:30
eIF5A promotes translation of Polyproline Motifs
Thomas Dever
NIH, United States of America
18
15:30 - 15:45
A proline triplet in Val-tRNA synthetase explains the origin
of EF-P and eIF5A
Agata Starosta
Gene Center Munich, LMU Munich, Germany
19
15:45 - 16:00
Molecular dynamics simulations of eukaryotic ribosomes:
integrating theory and experiment
Karissa Sanbonmatsu
Los Alamos National Laboratory and New Mexico
Consortium, United States of America
20
16:00 - 16:30
Coffee Break
ATC Foyer
16:30 - 16:45
The eEF2 kinase (eEF2K) promotes cell survival under
acute nutrient deprivation by blocking mRNA translation
elongation
Poul H Sorensen
BC Cancer Research Centre, Canada
21
16:45 - 17:00
Efficient -2 frameshifting by mammalian ribosomes
Andrew Firth
University of Cambridge, United Kingdom
22
Page 8
Programme
17:00 - 17:15
Jmjd4 is a lysyl hydroxylase of eRF1 required for optimal
translational termination
Mathew Coleman
University of Oxford, United Kingdom
23
17:15 - 17:30
Regulated stop codon readthrough provides a mechanism
for evolution of new gene functions
Joshua Dunn
University of California San Francisco, United States of
America
24
17:30 - 18:00
Refreshments
ATC Foyer
18:00 - 19:30
Dinner
ATC Canteen
19:30 - 21:30
Poster Session I
ATC Helices
21:30 - 23:00
Wine & Cheese and Meet the Speakers
ATC Foyer
Page 9
EMBO Conference Series: Protein Synthesis and Translational Control
Tuesday 10 September 2013
09:00 - 11:45
Session 3: Ribonucleoprotein Complexes
Chair: Jernej Ule
Klaus Tschira Auditorium
09:00 - 09:30
hiCLIP reveals the atlas of RNA structures recognized by
Staufen 1
Jernej Ule
UCL Institute of Neurology, United Kingdom
09:30 - 09:45
A role for Puf3 in oxidative stress tolerance in S. cerevisiae
26
Chris Kershaw
The University of Manchester, United Kingdom
09:45 - 10:00
New in vivo RNA-binding architectures discovered by
RBDmap
Alfredo Castello
EMBL Heidelberg, Germany
27
10:00 - 10:15
Oncogenic properties of the RNA-binding protein UNR:
Targets in melanoma progression
Laurence Wurth
Centre for Genomic Regulation, Spain
28
10:15 - 10:45 Coffee Break
ATC Foyer
10:45 - 11:00
Understanding the translational regulation and oncogenic
properties of Musashi 1 by integration of ribosome
profiling and target RNA immunoprecipitation
29
Scott Kuersten
Epicentre (An Illumina Company), United States of America
11:00 - 11:15
Poly(A) RNA and Paip2 act as allosteric regulators of
Poly(A)-binding protein
Seung Hwan Lee
POSTECH, Republic of Korea
Page 10
30
Programme
11:15 - 11:30
Enzymes in posttranscriptional control: investigating the
link between central metabolism and gene regulation
31
Benedikt Beckmann
EMBL Heidelberg, Germany
11:30 - 11:45
Glutamyl-prolyl tRNA synthetase is a novel
mTORC1-S6K1 effector that negatively regulates
translation of inflammation-related genes and determines
body mass
32
Paul Fox
Cleveland Clinic, United States of America
11:45 - 16:30
Free Time/ Sightseeing Downtown
16:30 - 19:15
Session 4: Mechanistic insights into initiation
Chair: Nicola Gray
Klaus Tschira Auditorium
16:30 - 17:00
DAZL regulates both the translation initiation and
deadenylation of germ cell mRNAs
Nicola Gray
University of Edinburgh, United Kingdom
33
17:00 - 17:15
Involvement of IF2 N domain in ribosomal subunit joining
revealed from architecture and function of the full-length
initiation factor
Angelita Simonetti
IGBMC, France
34
17:15 - 17:30
Mechanism of the initiator tRNA binding to the ribosomal
P-site in Escherichia coli
Umesh Varshney
Indian Institute of Science, India
35
17:30 - 17:45
Roles of eIF1 and 40S mRNA-exit channel protein Rps5e
in the response to start codon consensus sequence
36
Alan Hinnebusch
National Institutes of Health, United States of America
Page 11
EMBO Conference Series: Protein Synthesis and Translational Control
17:45 - 18:00
HCV-like IRESs sequester eIF3 to promote translation of
their viral mRNA
Amedee des Georges
HHMI-Columbia University, United States of America
37
18:00 - 18:30
Coffee Break
ATC Foyer
18:30 - 18:45
Histone H4 mRNA coding region contains a “Shine
Dalgarno-like” sequence allowing efficient translation
initiation
Franck Martin
IBMC - CNRS - UDS, Strasbourg, France
38
18:45 - 19:00
The tumor suppressor p53 acts as the safeguard of
translational control through direct regulation of fibrillarin
expression and rRNA methylation in cancer
39
Virginie Marcel
Cancer Research Center of Lyon, France
19:00 - 19:15
Translation acts upstream of transcription, in vivo
Daniela Brina
San Raffaele Scientific institute, Italy
19:15 - 20:30
Dinner
ATC Canteen
20:30 - 22:30
Poster Session II
ATC Helices
22:30 - 23:30
Wine & Cheese and Meet the Speakers
ATC Foyer
Page 12
40
Programme
Wednesday 11 September 2013
09:00 - 12:30
Session 5: The Ribosome
Chair: Roland Beckmann
Klaus Tschira Auditorium
09:00 - 09:30
Introduction + Structural basis of termination and
co-translational translocation in eukaryotes
Roland Beckmann
Gene Center Munich, Germany
41
09:30 - 09:45
Structural basis of signal sequence surveillance and
selection by the SRP-FtsY complex
Christiane Schaffitzel
EMBL Grenoble, France
42
09:45 - 10:00
Regulation of protein synthesis in bacterial dormancy by
reversible phosphorylation
Jonathan Dworkin (Presenter: Sandro Pereira)
Columbia University, Portugal
43
10:00 - 10:15
Mechanisms for efficient tRNA translocation
Christian Blau
MPI for Biophysical Chemistry, Germany
44
10:15 - 10:30
Mechanistic insight into ribosomal -1 frameshifting in
bacteria
Neva Caliskan
MPI for Biophysical Chemistry, Germany
45
10:30 - 11:00
Coffee Break
ATC Foyer
11:00 - 11:15
Small molecules on the eukaryotic ribosome
Nicolas Garreau de Loubresse
IGBMC, France
Page 13
46
EMBO Conference Series: Protein Synthesis and Translational Control
11:15 - 11:30
Peptidyl transferase inhibitors arrest ribosomes at specific
amino acid codons
Sergey Dmitriev
Belozersky Institute of Physico-Chemical Biology, Russian
Federation
47
11:30 - 11:45
Allosteric control of the Ribosome by small-molecule
antibiotics
Michael Wasserman (Presenter: Scott C. Blanchard)
Weill Cornell Medical College, United States of America
48
11:45 - 12:00
Gcn1 binding to the 40S ribosome head region is
essential for fully activating eIF2alpha kinase Gcn2.
Evelyn Sattlegger
Massey University, New Zealand
49
12:00 - 12:15
Structure of the ribosome•SelB complex at near-atomic
resolution
Niels Fischer
MPI for Biophysical Chemistry, Germany
50
12:15 - 12:30
Structural insights into drug-induced ribosomal stalling
51
Stefan Arenz
University of Munich, Germany
12:30 - 14:00
Lunch
ATC Foyer
14:00 - 16:00
Poster Session III
ATC Helices
16:00 - 18:15
Session 6: RNA Processing and Turnover
Chair: Oliver Mühlemann
Klaus Tschira Auditorium
16:00 - 16:30
Mechanistic aspects of NMD in human cells
Oliver Mühlemann
University of Bern, Switzerland
Page 14
52
Programme
16:30 - 16:45
How the interaction of PABPC1 with the translation
initiation complex inhibits nonsense-mediated decay of
transcripts with an AUG-proximal nonsense codon
53
Luísa Romão
Instituto Nacional de Saúde Dr. Ricardo Jorge, Portugal
16:45 - 17:00
The RQC complex in constant motion: from stalled 60S
recognition to aberrant nascent peptides degradation.
54
Quentin Defenouillère
Institut Pasteur, France
17:00 - 17:30
Coffee Break
ATC Foyer
17:30 - 17:45
Assembly and function of the CCR4-NOT complex
Elisa Izaurralde
MPI for Developmental Biology, Germany
55
17:45 - 18:00
The structural basis for the activity of a cytoplasmic RNA
uridylyltransferase
Luke Yates
University of Oxford, United Kingdom
56
18:00 - 18:15
Structural and functional analysis of the spliceosomal RNP
remodeling enzyme, Brr2
Karine Santos
Free University Berlin, Germany
57
18:15 - 19:15
Pre-Dinner Drinks & Meet the Speakers
ATC Foyer
19:15 - 01:00
Conference Dinner & Party
ATC Canteen and Foyer
Page 15
EMBO Conference Series: Protein Synthesis and Translational Control
Thursday 12 September 2013
09:15 - 12:00
Session 7: Non-coding RNA and Development
Chair: Antonio Giraldez
Klaus Tschira Auditorium
09:15 - 09:45
Using Ribosome footprinting to understand translational
regulation during vertebrate development
58
Antonio Giraldez
Yale University, United States of America
09:45 - 10:00 Designing a stress resistant translation machinery lessons from trypanosomatids
59
Michal Shapira
Ben-Gurion University of the Negev, Israel
10:00 - 10:15
A rice Cis-natural antisense RNA enhances PHO1;2
protein level via translational control and contributes to
phosphate homeostasis and plant fitness
60
Yves Poirier
University of Lausanne, Switzerland
10:15 - 10:30
tiRNAs assemble G-quadruplex structures to inhibit
translation initiation
Pavel Ivanov
Brigham nad Women's Hospital/Harvard Medical School,
United States of America
10:30 - 11:00
Coffee Break
ATC Foyer
11:00 - 11:15
miR-17-92 controls MYC-centered regulatory networks to
sustain growth of MYC-dependent lymphomas
Marija Mihailovic
European Institute of Oncology, Italy
Page 16
61
62
Programme
11:15 - 11:30
Interplay between microRNA-21 and RNA-binding protein
HuR in translation regulation of the pro-inflammatory
tumor suppressor gene Programmed Cell Death 4
(PDCD4
63
Partho Sarothi Ray
Indian Institute of Science Education and Research, India
11:30 - 11:45
RAN translation across intronic DM2 (CCTG) and ALS/FTD
(GGGGCC) expansion mutations
Laura Ranum
University of Florida, United States of America
64
11:45 - 12:00
System wide analyses have underestimated transcriptional
importance in animals
Mark Biggin
Lawrence Berekely Laboratory, United States of America
65
12:00 - 12:15
Closing Remarks
Klaus Tschira Auditorium
12:15 - 13:15
Packed Lunch & Departure
Page 17
EMBO Conference Series: Protein Synthesis and Translational Control
Page 18
Posters A-Z
Aeschimann, Florian
Dissecting the roles of GW182-interacting proteins in miRNA-mediated
gene repression
66
Andreev, Dmitry E.
Glycyl-tRNA synthetase is required for shifting Ribosome Landing Pad
to upstream AUG codon of PV IRES element
67
Antic, Sanja
mRNA degradation on the ribosome in Drosophila cells
68
Arenz, Stefan
Structural insights into drug-induced ribosomal stalling
69
Aviner, Ranen
Novel proteomic approach reveals cell cycle specific fluctuations in
mRNA Translation
70
Baggs, Eric
Elucidation of the determinants of IRES-mediated translation of cellular
mRNAs
71
Baranov, Pavel V.
Elucidating mechanisms of translation with computational analysis of
ribo-seq data
72
Bastide, Amandine
Post-transcriptional regulation of gene expression is essential for the
cellular response to cold stress
73
Bavli-Kertselli, Ira
Role of eIF5 phosphorylation in translation regulation
74
Biffo, Stefano
eIF6 activation and inhibition control tumorigenesis
75
Blanchet, Sandra
The natural suppressor tRNAs are differently incorporated during stop
codon readthrough in eukaryotes
Page 19
76
EMBO Conference Series: Protein Synthesis and Translational Control
Bojarska, Elzbieta
Substrate specificity of scavenger decapping enzymes (DcpS) towards
dinucleotide cap analogs modified within 7-methylguanine moiety
77
Brandon, Harland E.
Doing things differently: The universally conserved translational GTPase
HflX reveals a new mode of GTPase activation on the ribosome
78
Brook, Matthew
Characterisation of mammalian PABP4 expression and function
Burakovskiy, Dmitry E.
Impact of methylations of m2G966/m5C967 in 16S rRNA on bacterial
fitness and translation initiation
Cambiaghi, Tavane
Regulation of mammalian GCN2 by GCN1 and IMPACT
79
80
81
Cammas, Anne
HuR and miR-1192 respectively promote and reduce myogenesis by
modulating the translation of HMGB1 mRNA
82
Castellano, Mar M
General and differential changes in the translatome participate in the
establishment of the heat stress response in Arabidopsis seedlings
83
Castelli, Lydia M.
The translational repressors Caf20p and Eap1p are associated with
specific transcripts and actively translating ribosomes
84
Castello, Alfredo
New in vivo RNA-binding architectures discovered by RBDmap
Catez, Frederic
Impact of rRNA methyl-transferase Fibrillarin down-regulation on
ribosome synthesis and ribosome intrinsic activity
Page 20
85
86
Posters A-Z
Cencic, Regina
Modifiying chemotherapy response by targeted inhibition of eukaryotic
Initiation Factor 4A
87
Chew, Guo-Liang
Ribosome profiling reveals resemblance between long non-coding
RNAs and 5’ leaders of coding RNAs
88
Cockman, Matthew E.
OGFOD1 is a novel ribosomal prolyl hydroxylase involved in translational
control and stress granule formation
89
Cook, Simon J.
Modelling acquired resistance to mTOR kinase inhibitors
90
Cooke, Amy
Targeted identification and purification of TOP mRNA translational
regulators
91
Cowan, Joanne L.
The role and prevalence of non-canonical translation initiation codons in
generating mitochondrially-localised proteins
92
Curran, Joseph A.
RNAseq analysis of total and polysomal transcript populations in the
MCF7 and MCF10A cell lines demonstrates extensive cell-specific
mRNA 5’leader heterogeneity
93
Dassi, Erik
Genome-wide scanning for recurrent alterations of translation factors in
glioblastoma multiforme
94
Desnoyers, Guillaume
Investigating the mechanism of eIF3e-regulated
epithelial-to-mesenchymal transition
95
Draycheva, Albena G.
Interaction between the SRP receptor and the translocon SecYEG at
tme membrane
96
Page 21
EMBO Conference Series: Protein Synthesis and Translational Control
Duval, Mélodie
Ribosomal protein S1 unfolds structured mRNAs on the ribosome for
translation initiation in Escherichia coli
97
Epstein, Irina
Dissecting the message: Transcriptional and translational profiling of
mouse CA1 hippocampal neurons
98
Eskelin, Katri
Assaying plant ribosomes with asymmetric flow field-flow fractionation
99
Feng, Tianshu
Nucleolar proteins MINA53 and NO66 are ribosomal protein histidinyl
hydroxylases
100
Fieulaine, Sonia
Towards the structural characterization of co-translational action of
N-terminal methionine excision pathway enzymes
101
Firczuk, Helena
The ratio of eIF4G isoforms influences the balance between translation,
storage and degradation of mRNAs in Saccharomyces cerevisiae
102
Friday, Andrew J.
Spatial and temporal translational control of germ cell mRNAs by an
eIF4E Isoform, IFE-1
103
Fritsch, Claudia
Genome-wide search for novel human uORFs and N-terminal protein
extensions using ribosomal footprinting
104
Frolova, Ludmila
Two-step model of stop codon recognition by eukaryotic release factor
eRF1
105
Frost, Sigal
Upstream open reading frames (uORFs) in the 5'UTR of PKCeta
upregulate its expression during stress of high cell density
106
Page 22
Posters A-Z
Galicia Vazquez, Gabriela
Eukaryotic Initiation Factor 4AII is under MyoD control during
myogenesis
107
Gamm, Magdalena
The role of ribosomal heterogeneity in translational control by metabolic
signaling
108
García-Beyaert, Marina
Molecular mechanisms of SXL-mediated translational repression
109
Garreau de Loubresse, Nicolas
Small molecules on the eukaryotic ribosome
110
Geng, Songsong
Transmembrane Protein Coxsackievirus and Adenovirus Receptor (CAR)
associates with the translation machinery and regulates protein
synthesis
Glatt, Sebastian
Structural insights into Elongator function
111
112
Gorska, Agnieszka
The translational regulation of p53 and its deltaNp53 isoform using
antisense oligonucleotide strategy in vitro and in vivo
113
Grosso, Stefano
Expression and post translational modification of initiation factors
determines the response to chemotherapy in Malignant Mesothelioma.
114
Grzela, Renata
Characterization of different forms of peptide deformylases and their
interaction with bacterial ribosome
115
Haneke, Katharina
Translation control and stress granule formation during hypoxia
Hellen, Christopher U.
A novel mechanism of initiation mediated by the Halastavi árva RNA
Virus IRES
Page 23
116
117
EMBO Conference Series: Protein Synthesis and Translational Control
Heus, Hans A.
Insight into translation enhancers using ribosome profiling in Pichia
pastoris
118
Holtkamp, Wolf
Co-translational recruitment of SRP and SRP receptor to translating
ribosomes monitored in real-time
119
Horos, Rastislav
RNA-binding enzymes in hepatocytes
120
Iadevaia, Valentina
BOP1 mutant expression impairs ribosome biogenesis and activates
mTORC1 signalling
121
Itoh, Yuzuru
The decameric structure of SelA reveals the bacterial selenocysteine
formation mechanisms
122
Ivanova, Elena
Alu RNPs target 40S ribosomal subunits to repress translation initiation
123
Janich, Peggy
Analysis of the circadian translatome in mouse liver by ribosome
profiling
124
Kaija, Helena
Transcript profile of p21 gene and subcellular localization of the protein
in cardiac stress
125
Keiper, Brett D.
Translational control by initiation factors drives germ cell fate during
sperm and oocyte differentiation
126
Khan, Debjit
Translational control of p53 and ‘little-brother’ delta N-p53 in
glucose-deprivation
127
Khetchoumian, Konstantin
Molecular pathways for building a secretory cell
Page 24
128
Posters A-Z
Kiebler, Michael A.
The molecular mechanism of dendritic RNA localization in primary
neurons
Kimura, Yuichi
Pbp1 is involved in the Ccr4 and Khd1-mediated regulation of cell
growth through the association with ribosomal proteins, Rpl12a and
Rpl12b
Knight, John RP
Using the response to cold stress to analyse the role of SUMOylation in
RNA metabolism
Kotini, Suresh B.
Dynamics of selenocysteine incorporation in bacterial translation
129
130
131
132
Kubacka, Dorota
Investigating the consequences of eIF4E2 (4EHP) interaction with
4E-Transporter on its cellular distribution in HeLa cells
133
Kubick, Stefan
Orthogonal Biosystems: Cell-free synthesis of posttranslationally
modified membrane proteins
134
Kudrin, Pavel
Modifying the ppGpp scaffold: a molecular toolkit for investigation of the
stringent response
135
Kuhle, Bernhard
Purification and crystallization of the C-terminal domain of eukaryotic
translation initiation factor 5 (eIF5) in complex with an N-terminal
fragment of eIF2ß
136
Lacerda, Rafaela
Analysis of human Argonaute 1 5’ untranslated region shows internal
ribosome entry site activity
137
Lasfargues, Charline
Translational homeostasis through UPR-mediated massive expression
of 4E-BP1 in serous exocrine tissues
138
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EMBO Conference Series: Protein Synthesis and Translational Control
Lastdrager, Jeroen
Sucrose dependent stalling of ribosomes on the bZIP11 mRNA
139
Legen, Julia
Chloroplast Ribosome RNA association
140
Lehweß-Litzmann, Anja
Interaction of EF-G with L7/12 on the ribosome monitored in real time
using FRET
141
Liepelt, Anke
Translation control of TAK1 mRNA by hnRNP K modulates LPS-induced
macrophage activation
142
Lindqvist, Lisa M.
The translation initiation inhibitor silvestrol is highly selective for eIF4A
and induces cell death by multiple mechanisms
143
Liu, Yi
Expression, purification and crystallization of eukaryotic translation
initiation factor 3 subunit j(eIF3j/Hcr1)
144
Liu, Ying
Systematic analysis of transcription factors downstream of insulin
signaling
145
Lomakin, Ivan
Snapshots of the mammalian initiation of protein synthesis and the
mechanism of scanning
146
Lopez-Lastra, Marcelo
The 5’untranslated region of the human T-cell lymphotropic virus type 1
mRNA exhibits cap-independent translation initiation
147
Lopez-Lastra, Marcelo
The RNA chaperone protein Mex3A stimulates N-dependent translation
initiation of the ANDV SmRNA
148
Loreni, Fabrizio
Homeostatic regulation of ribosomal protein synthesis involves changes
in translation elongation
149
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Posters A-Z
Lukash, Tetyana O.
Identification and comparative analysis of in vivo phosphorylation of
eEF1A1 and eEF1A2
Lukaszewicz, Maciej R.
Decapping activity of human Nudt16 enzyme towards short
ribooligonucleotides capped with novel monomethylated (MMG) and
trimethylated (TMG) cap analogs modified in the triphosphate chain with
S or NH.
Macdonald, Paul
cis regulatory elements of oskar mRNA regulate translation in trans
Mallucci, Giovanna
The role of the unfolded protein response in neurodegeneration: a new
target for therapy of these disorders
150
151
152
153
Malys, Naglis
Exploring role of translation initiation in stochastic gene expression
154
Mancera-Martinez, Eder
Reinitiation-supporting protein RISP interacts with eIF3 and eIF2
155
Marques-Ramos, Ana
Internal ribosome entry site-mediated translational regulation of
mammalian target of rapamycin (mTOR) in stress conditions
156
Martinez-Salas, Encarna
Involvement of the RNA-binding protein Gemin5 in IRES-dependent
translation
157
Mechulam, Yves
Structure of the archaeal translation initiation complex
158
Merrick, William C.
Influence of translation factor activities on start site selection
159
Michel, Audrey M.
GWIPS-viz: Development of a dedicated ribo-seq genome browser
160
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EMBO Conference Series: Protein Synthesis and Translational Control
Mikhaylova, Tatiana
Human Dbp5 regulates translation termination activity of eRF1
161
Millevoi, Stefania
Translationally silent but tuneable G-quadruplex structure at the VEGF
IRES-A
162
Mohammad-Qureshi, Sarah S.
A dependancy on eIF2B Phosphorylation for cell cycle progression in
stressed yeast
163
Moore, Kat
Selective mRNA translation controls erythropoiesis: regulation and
function of the RNA-binding protein Csde1
164
Morrison, J. Kaitlin
The role of cap-independent mRNA translation in germ cell fate
decisions
165
Müller, David
4E-BP restrains eIF4E phosphorylation
166
Munoz, Antonio M.
The role of initiator tRNA structure in transmitting the start codon
recognition signal
167
Mureev, Sergey
Anticodon-specific replacement of tRNA isoacceptors by their
orthogonal counterparts for site-selective protein labeling in vitro
168
Nagarajan, Sabarish
Nutrition dependent regulation of mRNA translation during Drosophila
growth and development
169
Namy, Olivier
Genome-wide translational consequences of the yeast prion [PSI+]
Page 28
170
Posters A-Z
Nedialkova, Danny
tRNA wobble uridine hypomodification disrupts protein homeostasis by
decreasing the decoding efficiency of cognate codons in vivo
171
Niepmann, Michael
microRNA-122 dependent binding of Ago2 protein to Hepatitis C Virus
RNA associated with enhanced RNA stability and translation stimulation
172
Noeske, Jonas
High-resolution structural analysis of solvent and ion interactions with
the ribosome
173
Nousch, Marco
Charaterization of polyadenylation and deadenylation in the C. elegans
germ line
174
O'Brien, Edward
Understanding cotranslational protein folding at the molecular and
cellular levels using theory and computation
175
Ohlmann, Theo
Molecular insights into the effect of miRNAs on translation repression
and stimulation
176
Olsthoorn, René C.
Stimulation of ribosomal frameshifting by RNA G-quadruplex structures
177
Osterman, Ilya
Bacterial mRNA features affecting translation initiation and reinitiation –
in vivo and vitro studying
178
Pandey, Poonam
Understanding the mechanism of differential regulation of insulin gene2
splice variants
179
Panthu, Baptiste
In vitro veritas: an adaptable mammalian cell free system that
reproduces physiological cellular conditions for in vitro translation
180
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EMBO Conference Series: Protein Synthesis and Translational Control
Panthu, Baptiste
The NS1 protein from influenza A is a general enhancer of the host
translation
181
Perry, Lisa
Maintaining fidelity? A glimpse into the rules governing mammalian start
codon selection
182
Pichon, Xavier
Cytoplasmic polypyrimidine tract binding protein controls mRNAs that
regulate cytoskeletal organisation and cell migration
183
Piñeiro, David
Do EJCs enhance L-myc IRES dependent translation?
Polunovsky, Vitaly
eIF4E-mediated translational control of the stem/progenitor cell
transition to malignancy: Implications for pregnancy-associated breast
oncogenesis
Preiss, Thomas
RBP atlas: an exploration of interactions between mRNA and proteins
and their impact on cardiomyocyte biology
Preiss, Thomas
miRNA profiles characterise distinct states of cellular pluripotency
Preiss, Thomas
Shifting targets: microRNA variants and alternative polyadenylation in
cardiac hypertrophy
Preiss, Thomas
A tale of two termini: profiling mRNA 5'-3' interactions in vivo
184
185
186
187
188
189
Preiss, Thomas
RNA methylation: a mechanism for post-transcriptional regulation that is
deregulated in cancer?
190
Provenzani, Alessandro
A novel high throughput biochemical assay to evaluate HuR-RNA
complex formation
191
Page 30
Posters A-Z
Radhakrishnan, Aditya
Exploring the role of Dhh1-Ribosome interactions in general translation
repression
192
Rajman, Marek
Homestatic plasticity - paradigm to study regulation of protein
translation by microRNAs
193
Rorbach, Joanna
Characterisation of novel methyltransferases involved in modifications of
mitochondrial large subunit rRNA
194
Rossi, Danuza
eIF5A binds directly to the 60S ribosomal subunit
Ruzzenente, Benedetta
NSUN4 is a bi-functional methyltranferase required for the biogenesis of
the mitochondrial ribosome
Sachs, Matthew
Conserved non-AUG translation initiation in the mRNA of cpc-1 from
Neurospora crassa suggests potential for previously unrecognised layer
of regulation
Saguy, Matthieu
Visualisation of +1 frameshifting during tRNA accommodation
Savulescu, Anca
High content screening approach to identify translational regulators of
RNA transport
195
196
197
198
199
Saxena, Manisha
Characterization of Yeast eIF4E post-translational modification
200
Scheckel, Claudia
nELAVL mediated RNA regulation during Alzheimer's Disease
201
Schibich, Daniela
Proteome-wide analysis of the nascent chain interactome of the signal
recognition particle by selective ribosome profiling
Page 31
202
EMBO Conference Series: Protein Synthesis and Translational Control
Schmid, Tobias
IRES-dependent translation of cyp24A1 is controlled by PI3K-Akt
signaling
Schofield, James
A G-quadruplex within the 5’ UTR of the acid-sensitive potassium leak
channel, TASK-3, determines mRNA fate and membrane expression of
the channel
203
204
Schott, Johanna D.
Translational control of specific mRNAs is important for cellular survival
and the anti-inflammatory feedback during macrophage activation
205
Schulz, Julia
Translational control of proto-oncogene expression by upstream open
reading frames (uORFs)
206
Schuster, Birgit
RNP capture of defined RNA species in vivo
207
Schwarz, Juliane
A systems analysis of translation in totipotent stem cells
208
Sekirnik, Rok
Ribosomal protein hydroxylation is a new post-translational modification
of translational machinery conserved from prokaryotes to humans
209
Shapira, Michal
Designing a stress resistant translation machinery - lessons from
trypanosomatids
210
Sidarovich, Viktoryia
Validation of a cell-based high-throughput screening assay for
posttranscriptional regulation
211
Sidrauski, Carmela
Comprehensive profiling of the eIF2a-mediated unfolded protein
response
212
Page 32
Posters A-Z
Simpson, Clare E.
Characterization of the eIF4E-binding protein, 4E-T, in mammalian cells
213
Smirnova, Victoria V.
The mechanism of translation initiation of the unspliced HIV-1 mRNA
214
Smith, Ewan M.
Investigation of the translational regulation of terminal oligo pyrimidine
tract (TOP) containing mRNAs
215
Sohmen, Daniel
Structural basis for species-specific polypeptide-mediated translational
arrest
216
Sokolova, Elizaveta
The influence of stop codon 3'-context on competition between
termination factors and supressor tRNAs
217
Standart, Nancy
4E-T represses translation of tethered mRNAs in a P-body- and eIF4Eindependent manner, and enhances silencing of microRNA-target
mRNAs
Stanhill, Ariel
eIF1 phosphorylation mediates leaky scanning translation initiation
218
219
Stewart, Joanna D.
ABC50 plays a critical role in start-site selection during mRNA
translation
220
Stoneley, Mark
A proteomic analysis of changes in the RNA binding protein interactome
during chemotherapeutic stress
221
Sträßer, Katja
The phosphoproteome of ribosomes
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EMBO Conference Series: Protein Synthesis and Translational Control
Sundaram, Arunkumar
Regulation of translation initiation during stress conditions in Candida
albicans
223
Svetlov, Maxim S.
Erythromycin enhances the naturally occurring Peptidyl-tRNA drop-off
from the translating ribosome
224
Swiatkowska, Agata
The p53 translation initiation and the role of potential IRES element in
this process
225
Tebaldi, Toma
tRanslatome: an R package to portray translational controls hidden in
high-throughput assays
226
Terenin, Ilya M.
One mRNA, two mechanisms. The case of HCV IRES.
Therizols, Gabriel
5-fluorouracil treatment of colorectal cancer cells alters ribosome
biogenesis and cytoplasmic ribosomal RNA composition
227
228
Thiadens, Klaske A.M.H.
Selective mRNA translation in erythropoiesis
229
Tieg, Bettina
Analyses of the translation termination factors in S. cerevisiae
230
Urbanska, Anna
Regulation of dendritogenesis by ZBP1 depends on its phosphorylation
at Ser181
231
von der Haar, Tobias
Translation elongation controls translation initiation on eukaryotic
mRNAs
232
von Loeffelholz, Ottilie
Structural basis of signal sequence surveillance and selection by the
SRP–FtsY complex
233
Page 34
Posters A-Z
Vopalensky, Vaclav
Cap-independent translation of mRNAs encoded by yeast linear
plasmids
Wethmar, Klaus
uORFdb – a comprehensive literature database on uORF biology
234
235
Wieden, Hans-Joachim
Identification of conserved intramolecular communication pathways
within EF-Tu
236
Wortham, Noel
Dimerisation of the eIF2B complex, a potential new mechanism for
translational control?
237
Yanagiya, Akiko
Translational control of IFN-beta mRNA via RNA-binding proteins
238
Yerlikaya, Seda
Regulation and function of Rps6 Phosphorylation in budding yeast
239
Yoffe, Yael
DAP5 mediated translation control of human embryonic stem cell
differentiation
Zach, Lolita
eIF1 phosphorylation mediates leaky scanning translation initiation
240
241
Zamudio Ochoa, Angelica Raquel
The role of Pet309 in mitochondrial translation initiation of the COX1
mRNA
242
Zanelli, Cleslei
eIF5A has a function in the cotranslational translocation of proteins into
the ER
243
Zielonka, Elisabeth M
System-wide posttranscriptional responses of NIH-3T3 cells to
genotoxic stress
244
Page 35
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Speaker Abstracts
1
A historical perspective: translation initiation in eukaryotes in health
and disease
Nahum Sonenberg
McGill University, Canada
Presenter: Nahum Sonenberg
In the past 40 years, we have witnessed remarkable advances in the understanding of the
mechanisms of translation initiation in eukaryotes, starting with the identification and
characterization of initiation factors and the discovery of the mRNA cap structure, and the poly
A tail. These were followed by the discovery of the IRES (internal ribosome entry site) and
uORFs (upstream open reading frames), and their physiological importance. The mechanistic
knowledge led to experiments to understand the control of translation initiation, such as in
learning and memory and during stress, and its dysregulation in diseases such as cancer,
autism and viral infections. I will discuss some of these discoveries from a personal
perspective.
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EMBO Conference Series: Protein Synthesis and Translational Control
2
Structural studies of eukaryotic ribosomes and functional insights
Nenad Ban
ETH Zurich, Switzerland
Presenter: Nenad Ban
To be announced
Page 38
Speaker Abstracts
3
microRNA mechanism: Are we there yet?
Martin Bushell
MRC Toxicology Unit, United Kingdom
Presenter: Martin Bushell
MicroRNAs (miRNAs) are non-coding 21-25 nucleotide RNA molecules that in metazoans
base-pair imperfectly with regions in target mRNAs (generally within the 3’UTR) and repress
the synthesis of the corresponding proteins. The mechanism for miRNA-mediated repression
has remained elusive. Nevertheless, it is clear that miRNAs bind target mRNAs in complex with
Argonaute proteins (Ago1-4 in humans). This complex then recruits one of the trinucleotide
repeat containing proteins (TNRC6A-C in humans) and in turn leads to both translational
repression and mRNA destabilisation. However, the interplay between these processes and
the precise molecular mechanisms involved remain unclear. Our recent data suggests that
translational inhibition is the critical process for miRNA-mediated repression while mRNA
deadenylation and mRNA degradation are secondary effects which are not required.
Translational inhibition depends on miRNAs impairing the function of the eIF4F initiation
complex. We define the RNA helicase eIF4A2 as the key factor of eIF4F through which
miRNAs function. Importantly, here we show by MS of endogenously purified eIF4A2 and
eIF4A1 complexes that cNOT1 (CCR4-NOT complex component) interacts strongly with only
eIF4A2. We uncover a correlation between the presence of miRNA target sites in the 3’UTR of
mRNAs and secondary structure in the 5’UTR, and show that mRNAs with unstructured
5’UTRs are refractory to miRNA repression. These data support a linear model for miRNAmediated gene regulation in which translational repression via eIF4A2 is required first, followed
by mRNA destabilisation.
Page 39
EMBO Conference Series: Protein Synthesis and Translational Control
4
eIF2B displaces eIF5 from eIF2•GDP to promote efficient nucleotide exchange
and activation of eIF2
Martin Jennings, Yu Zhou, Graham Pavitt
University of Manchester, United Kingdom
Presenter: Martin Jennings
During translation initiation, eIF2•GTP delivers initiator tRNA to the 40S ribosome. The
G-protein cycle of eIF2 is critical for this function with each round of deactivation and
reactivation driving successive translation events. Two proteins regulate eIF2’s G-protein cycle:
eIF5 has both GTPase accelerating protein (GAP) and GDP dissociation inhibitor (GDI)
functions, and eIF2B is the guanine nucleotide exchange factor (GEF). This process of eIF2
reactivation by eIF2B is critical for viability, with mutations in eIF2B causing the fatal brain
disease VWM (Leukoencephalopathy with vanishing white matter). Furthermore this step is
crucial for translation regulation where eIF2B is inhibited by eIF2(α) phosphorylation. eIF2B is a
particularly complex GEF, being ~300 kDa and comprising five separate subunits. Recent
findings that eIF2 is bound to eIF5 before reactivation where eIF5 exhibits nucleotide
stabilization (GDI activity) has led us to question how eIF2B accesses eIF2 to permit nucleotide
exchange. We have used protein-protein interaction and nucleotide exchange assays to
monitor the kinetics of eIF2 release from the eIF2•GDP/eIF5 GDI complex and determine
eIF2B’s effect on this release. We demonstrate that eIF2B has a second activity as a GDI
displacement factor (GDF) that can recruit eIF2 from the eIF2•GDP/eIF5 GDI complex prior to
GEF action. This explains how eIF2B can compete effectively with eIF5 for eIF2 binding. We
find that GDF function is dependent on the catalytic sub-complex eIF2Bγε. Furthermore GDF is
not inhibited when eIF2α is phosphorylated, demonstrating that GDF and GEF activities are
independent. Finally, we show that an eIF2Bγ mutant affecting cell growth specifically impairs
GDF activity. This demonstrates an in vivo requirement for this new eIF2B function and raises
the possibility that GDF function may play a role in some VWM disease mutations and
translational control of GCN4.
Page 40
Speaker Abstracts
5
eIF4G and eIF1 cooperate to facilitate translation start codon selection
Rivka Dikstein1, Ora Haimov1, Hadar Sinvani1, Yuri Svitkin2, Nahum Sonenberg2
1 The Weizmann Institute of Science, Israel
2 McGill University, Canada
Presenter: Rivka Dikstein
The translation initiation factors eIF4G and eIF1 are known to play distinct roles in translation
initiation. eIF4G promotes mRNA cap recognition and 5’UTR scanning through eIF4E and
eIF4A, while eIF1 directs AUG recognition or bypass in a manner dependent on the AUG
context and the length of the 5’UTR. Presently little is known about the potential involvement of
eIF4G in start codon selection. Here we investigated the role of these eIFs in translation of
mRNAs with AUG in various contexts and 5’UTR length. Our data reveal a surprising role of
eIF4G in translation initiation fidelity. Downregulation of eIF4G prevented leaky scanning and
enhanced initiation from short 5’UTR mRNA reminiscent of the effect observed upon loss of
eIF1. Remarkably, the opposite effect is observed with short 5’UTR bearing the Translation
Initiator of Short 5’UTR (TISU) element, as initiation is abolished in the absence either eIF4G or
eIF1. We further demonstrate and this functional cooperation between eIF4G and eIF1 is
facilitated by direct and specific interaction between these factors. Although yeast and human
eIF1 are more than 90% identical, the yeast protein fails to promote leaky scanning in human
cells and to interact with eIF4G. Our results provide insight into the molecular basis and
evolution of AUG-context dependent translation and identify eIF1 and eIF4G as principal
players.
Page 41
EMBO Conference Series: Protein Synthesis and Translational Control
6
CLIP-seq analysis of the eIF4F complex reveals principles of mRNP formation
and regulation
David Weinberg, David Bartel
Whitehead Institute / MIT, United States of America
Presenter: David Weinberg
In eukaryotes, translating mRNPs adopt a closed-loop conformation in which the 5′ and 3′
ends are bridged by protein factors, enhancing translation and mRNA stability. The central
component of the closed-loop mRNP is eIF4G, which simultaneously interacts with the
cap-binding protein eIF4E and poly(A)-binding protein (PABP). Previous analyses of the closed
loop have been limited by a lack of suitable assays to study this and other aspects of
translation initiation in vivo. Here we apply CLIP-seq (crosslinking and immunoprecipitation
followed by sequencing) analysis of translation factors to study the translating mRNP in vivo.
We find that yeast eIF4G directly interacts with both mRNA ends in vivo, which provides a
global assay for mRNA circularization. Distinct RNA-binding regions interact preferentially with
the 5′ and 3′ ends, consistent with a single eIF4G molecule bridging both mRNA ends.
Surprisingly, disrupting the eIF4G–PABP interaction reduces but does not eliminate 3′-end
binding by eIF4G, suggesting that additional factors contribute to the 3′-end localization of
eIF4G. We identify one such factor that interacts directly with both eIF4G and the mRNA 3′
end, thereby enhancing eIF4G binding to the 3′ end in the absence of an eIF4G–PABP
interaction. Moreover, our analyses provide insight into the wide range of translational
efficiencies (TEs) observed for endogenous mRNAs, the molecular basis of which has
remained enigmatic. By extending our CLIP-seq analyses to the entire eIF4F cap-binding
complex (comprised of eIF4G, eIF4E, and the RNA helicase eIF4A), we find that eIF4F binding
accounts for at least half of the variance in TE. These results suggest that TE is determined
largely at the first step of cap-dependent translation (mRNA recognition by the eIF4F complex),
before ribosome recruitment and scanning.
Page 42
Speaker Abstracts
7
Positional proteomics and ribosome profiling to map the translation initiation
landscape
Petra Van Damme, Daria Gawron, Gerben Menschaert, Wim Van Criekinge, Kris Gevaert
University of Ghent, Belgium
Presenter: Petra Van Damme
Alternative usage of presumed 5’UTR or downstream, in-frame AUG codons, in addition to
non-AUG codons as translation start codons, contributes to the diversity of a proteome as
protein isoforms harboring different N-terminal extensions or truncations can serve different
regulatory mechanisms. Recent ribosome profiling data revealed a highly underestimated
occurrence of database non-annotated, and thus alternative translation events (aTIS) at the
mRNA level. N-terminomics data revealed that in higher eukaryotes more than 10% of all
identified protein N-termini point to aTIS, to incorrect assignments of the translation start
codon, translation initiation at near-cognate start codons, or to alternative splicing. We here
report on more than 2,000 unique alternative protein N-termini identified in human or mice at
the proteome level. Customized databases created using the translation initiation mapping
obtained from ribosome profiling data additionally demonstrates the usage of initiator
methionine encoding near-cognate start codons besides the existence of N-terminal extended
protein variants at the level of the proteome. Various newly identified aTIS were confirmed
using mutagenesis studies, and meta-analyses demonstrated that aTIS reside in strong
Kozak-like motifs and are conserved among eukaryotes, hinting to their biological importance.
Finally, TargetP analysis predicted that the usage of aTIS often results in altered subcellular
localization patterns, thus hinting to their functional diversification.
Page 43
EMBO Conference Series: Protein Synthesis and Translational Control
8
mTORC1-regulated translation of the C/EBPß-mRNA determines health- and
life-span in mice
Laura Zidek, Christine Müller, Götz Hartleben, Sabina Eichwald, Cornelis Calkhoven
Leibniz Institute for Age Research - Fritz Lipmann Institute, Germany
Presenter: Cornelis Calkhoven
Improvement of metabolic health as can be achieved by caloric restriction (CR) extends health
and lifespan. However, following a strict CR feeding regime is rather difficult to achieve for
humans. Therefore, pathways and their downstream effectors need to be identified that
improve metabolism independently from CR feeding regimes. While inhibition of the mTORC1
pathway results in metabolic improvements similar to CR, the downstream regulatory factors
that mediate these effects are still unknown. Translation of the C/EBPβ mRNA into the
C/EBPβ-LIP protein isoform is achieved through re-initiation at a downstream AUG-codon
following initial translation of an upstream open reading frame (uORF). Activation of mTORC1
promotes C/EBPβ-LIP translation, which is also strongly induced upon loss of 4E-BP1 and
4E-BP2. In contrast, pharmacological inhibition of mTORC1 or knockdown of the
mTORC1-specific component raptor, but not of the mTORC2-specific component rictor,
results in suppression of C/EBPβ-LIP. We have generated uORF-deficient (ΔuORF) knock-in
mice that fail to translationally up-regulate C/EBPβ-LIP and therefore mimic low mTORC1
activity at the level of C/EBPβ translation. These mice recapitulate beneficial metabolic
phenotypes that are hallmarks of CR and reduced mTORC1 signalling, including increased
fatty metabolism, insulin sensitivity and glucose tolerance. In addition, C/EBPβ-ΔuORF mice
display reduced somatotropic growth hormone (GH)/ IGF-1 signalling, features known to
extend health- and life-span in mice. In an on-going lifespan determination experiment survival
of female wt mice has reached median value (25✝ of 50) whilst the mortality of female
C/EBPβ-ΔuORF mice is significantly lower (9✝ of 50), indicating an increase in lifespan for the
latter. Thus, changed translation of a single mTORC1 target mRNA - the C/EBPβ-mRNA - is
sufficient to extend health-span and most likely lifespan.
Page 44
Speaker Abstracts
9
An upstream open reading frame in ERCC5 establishes resistance to Cisplatin
Joanna Somers1, Ian G. Cannell2, John-Paul Kilday3, Lindsay A. Wilson1, Emilie Horvilleur1,
Tuija Pöyry1, Laura Cobbold4, Alexander Kondrashov5, Celine Ferreira6, Stephanie Puget7,
Jacques Grill8, Richard G. Grundy3, Martin Bushell1, Anne E. Eillis1
1 MRC Toxicology Unit, United Kingdom
2 David H. Koch Institute for Integrative Cancer Research, MIT, United States of America
3 Children’s Brain Tumour Research Centre, University of Nottingham, United Kingdom
4 University of Nottingham, United Kingdom
5 School of Pharmacy, University of Nottingham, United Kingdom
6 Institut Gustave Roussy, France
7 Hôpital Necker, University Paris V Descartes, France
Presenter: Joanna Somers
ERCC5 is a DNA damage repair protein in the nucleotide excision repair pathway, responsible
for the removal of bulky DNA lesions caused by platinum-chemotherapeutics. Here we show
that a common single nucleotide polymorphism (rs751402, G/A) in the 5' UTR of ERCC5
creates an uORF that determines sensitivity to platinum-based chemotherapy by controlling
ERCC5 translation. We demonstrate in both normal and cancer human cell lines that
endogenous ERCC5 expression after cisplatin treatment is uORF dependent (maintained in
A/A, A/G, rapidly decreased in G/G), correlating with the marked resistance of A/A and A/G
cells to cisplatin. We confirm that this occurs at the level of translation and following treatment
of cells with cisplatin there was a greater retention of the uORF/A transcript in the polysomes.
Moreover, sensitivity to cisplatin was restored by addition of a DNA-PKcs inhibitor, indicating
that DNA-PKcs is required to maintain ERCC5 translation. Importantly, we show that the
rs751402 genotype is an independent prognostic marker for progression-free survival after
platinum-based chemotherapy in paediatric Ependymoma patients and the presence of the
uORF/A transcript significantly reduced progression-free survival (multivariate hazard ratio 3.63,
95% CI 1.69-7.76). Our data support a model whereby the A but not the G transcript of
ERCC5 contains a functional uORF, that attenuates translation under normal conditions but
which maintains translation when overall protein synthesis is inhibited. We conclude that
screening of Ependymomas (and potentially other tumours) a priori for the rs751402 can
predict those who will respond to platinum-based therapy and that DNA-PKcs inhibitors could
provide alternative therapeutic options.
Page 45
EMBO Conference Series: Protein Synthesis and Translational Control
10
CGG repeat associated non-AUG initiated translation
Seok Yoon Oh1, Amy Krans1, Fang He1, Chantal Sellier2, Nicholas Charlet2, Peter Todd1
1 University of Michigan, United States of America
2 IGBMC, France
Presenter: Peter Todd
Fragile X-associated Tremor Ataxia Syndrome (FXTAS) is a neurodegenerative illness that
results from a CGG repeat expansion in the 5’UTR of the FMR1 gene. We have recently shown
that these CGG repeats are capable of supporting translational initiation within the 5'UTR via
an AUG independent mechanism. This “RAN” translation contributes directly to inclusion
formation and neurodegeneration in model systems. Surprisingly, initiation is detected in two of
three possible reading frames relative to the repeat, and the constraints on initiation differ
depending on the reading frame used. The major translation product, FMRpolyG, is a
polyglycine containing protein whose translation initiates at a near-AUG codon just 5' to the
repeat and which is sensitive to interventions which impact cap-dependent translation. In
contrast, translational initiation in a different reading frame produces a polyalanine protein
(FMRpolyA). This process is less efficient, occurs only with larger repeat expansions, and
appears to occur within the repeat itself in the absence of any near-AUG codon. These
findings suggest that repetitive RNA elements may impact on translational initiation site choices
and thereby contribute to proteome diversity and human disease.
Page 46
Speaker Abstracts
11
Cellular mRNAs access second ORFs using a novel amino acid
sequence-dependent coupled translation termination-reinitiation mechanism
Phillip Gould, Nigel Dyer, Sascha Ott, Andrew Easton
University of Warwick, United Kingdom
Presenter: Phillip Gould
Polycistronic transcripts are considered rare in the human genome. Initiation of translation of
internal ORFs of eukaryotic genes has been shown to use either leaky scanning or highly
structured IRES regions to access initiation codons. Studies on mammalian viruses identified a
mechanism of coupled translation termination-reinitiation that allows translation of an additional
ORF. Here, the ribosome terminating translation of ORF-1 translocates upstream to reinitiate
translation of ORF-2. We have an algorithm to identify mRNAs in the human transcriptome in
which the major ORF-1 overlaps a second ORF capable of encoding a product of at least 50aa
in length. This identified 4368 transcripts representing 2214 genes. We investigated 25
transcripts, 23 of which were shown to express a protein from ORF-2 highlighting that 3’UTRs
contain protein coding potential more frequently than previously suspected. Six transcripts
accessed ORF-2 using a process of coupled translation termination-reinitiation. Analysis of one
transcript, encoding the CASQ2 protein, showed that the mechanism by which the coupling
process of the cellular mRNAs was achieved was novel. This process was not directed by the
mRNA sequence but required an aspartate rich repeat region at the C-terminus of the
terminating ORF-1 protein. Introduction of wobble mutations for the aspartate codon had no
effect whereas replacing aspartate for glutamate repeats eliminated translational coupling. This
is the first description of a coordinated expression of two overlapping proteins from cellular
mRNAs using a coupled translation termination-reinitiation process and is the first example of
such a process being determined at the amino acid level.
Page 47
EMBO Conference Series: Protein Synthesis and Translational Control
12
TOR signaling regulates reinitiation of translation
Mikhail Shchepetilnikov, Lyubov Ryabova
Université de Strasbourg, France
Presenter: Mikhail Shchepetilnikov
Aberrant regulation of translation initiation in eukaryotes causes crucial abnormalities in
developmental programs. The evolutionary conserved target of rapamycin (TOR) kinase
promotes cell growth and metabolism in response to environmental stimuli via phosphorylation
of eIF4E-binding proteins (4E-BPs) and 40S RPS6 kinase 1 (S6K1) that positively affects
general cap-dependent translation initiation. In contrast, translational regulation of the
alternative initiation mechanism, i.e. reinitiation after upstream ORF (uORF) translation, has not
been yet clarified. However, expression of many growth and transcription factors is under
control of multiple uORFs in 5'-UTR of their encoded mRNAs. Mutations affecting uORFs can
provoke expression of potential pro-oncogenic factors and reprogramming of the whole
translational landscape in cells. We demonstrated the functional TOR pathway in plants, where
hormone auxin can trigger TOR activation followed by S6K1 phosphorylation at T449 and
promote reinitiation after uORF translation. When activated, TOR binds polysomes stimulating
reinitiation capacity of translating ribosomes. Indeed, selected uORF-mRNAs were found
preferentially associated with polysomes. We identified subunit h of eIF3 (eIF3h) as a
downstream target of TOR signaling. Many cancer cells accumulate high levels of eIF3h, where
its oncogenic potential is increased by phosphorylation at S183. Strikingly, phosphorylation of
eIF3h up-regulates reinitiation after uORF translation in plants, and reinitiation is abolished in
mutant plants expressing defective eIF3h. According to above data eIF3h seems to be a
unique reinitiation promoting factor (RPF) that is critical for translation of uORF-containing, but
dispensable for translation of monocistronic mRNAs. In accordance, TOR activation correlates
with recruitment and phosphorylation of eIF3h at S178 in polyribosomes. Our recent data on
the role of eIF3h in translation reinitiation will be presented.
Page 48
Speaker Abstracts
13
Functional profiling of IRES elements during cellular hypoxia by translation of
circular RNAs
Eric Mills1, Nicholas Ingolia2
1 Johns Hopkins University School of Medicine, United States of America
2 Carnegie Institution for Science, United States of America
Presenter: Eric Mills
Internal ribosome entry sites (IRESs) recruit ribosomes to mRNAs directly and promote their
translation independent of the 5’ mRNA cap. During hypoxia, when canonical cap-dependent
translation is repressed, IRESs are proposed to maintain expression of stress-responsive
mRNAs, and thereby serve as cis-acting regulatory sequences controlling a broad
post-transcriptional expression program. Our understanding of this regulatory program has
been limited by the technical challenges of characterizing IRES elements, particularly in cellular
(rather than viral) transcripts. To address these questions, we describe IRESeq, a novel
high-throughput strategy for functionally profiling IRES activity based on the ability of
sequences derived from human transcriptomic fragments to stimulate translation of a
covalently circular RNA reporter. We first validated this strategy using cellular and viral IRES
sequences. We then profiled IRES activity in reporter libraries containing >107 unique
fragments of the human transcriptome and identified novel candidate IRESs in normoxic and
hypoxic cells.
Page 49
EMBO Conference Series: Protein Synthesis and Translational Control
14
Mechanisms of eukaryotic reinitiation
Tatyana Pestova
SUNY Downstate Medical Center, United States of America
Presenter: Tatyana Pestova
During ribosome recycling, post-termination complexes (post-TC) are dissociated by ABCE1
and eRF1 into 60S and tRNA/mRNA-associated 40S subunits. Subsequent dissociation of
tRNA can be promoted by eIFs 1/1A, Ligatin, or MCT1/DENR. tRNA release is followed by
mRNA dissociation. At low [Mg2+], the entire ribosome recycling can also be mediated by eIFs
3/1/1A, with eIF3 being responsible for splitting of post-TCs into subunits. However, in some
cases post-TCs do not undergo complete recycling: 40S subunits remain bound to mRNA,
and termination is followed by reinitiation, usually downstream of the stop codon. To
investigate the mechanism of reinitiation, we recapitulated it in vitro using a mammalian
reconstituted translation system. We found that when ABCE1-dependent or independent
splitting of post-TCs proceeds in the presence of Met-tRNAiMet and eIFs 2/3/1/1A, 40S
subunits remain bound to mRNA and efficiently reinitiate at nearby upstream and downstream
AUGs. However, eIFs 2/3/1/1A cannot impose 3’-directionality on scanning recycled 40S
subunits, and formation of 48S complexes at either upstream or downstream AUGs is
determined by the lack of secondary structure in the corresponding region between the AUG
and the stop codon. 3’-directionality of reinitiation is promoted by the group 4 eIFs. We also
found that that post-termination ribosomes are not stably anchored on mRNA, and can
migrate to nearby upstream and downstream codons that are cognate to the P-site deacylated
tRNA. The efficiency of migration depends on the mode of peptide release
(puromycin>eRF1/eRF3) and the nature of the P-site tRNA (e.g. tRNACys >tRNALeu). eEF2
strongly promotes migration and also destabilizes association of post-termination ribosomes
with eRF1, whereas E-site tRNA and elevated [Mg2+] both reduce migration and stabilize
ribosomal binding of eRF1. The mobility of post-termination ribosomes suggests that some
reinitiation events could involve 80S ribosomes rather than 40S subunits.
Page 50
Speaker Abstracts
15
Accuracy of decoding on the ribosome is maintained by insisting on
Watson-Crick geometry
Natalia Demeshkina1, Alexey Rozov1, Lasse Jenner2, Marat Yusupov1, Gulnara Yusupova1
1 IGBMC, France
2 University of Aarhus, Denmark
Presenter: Natalia Demeshkina
One of the fundamental questions in molecular biology is how the ribosome selects correct
(cognate) tRNAs from a large pool of incorrect (near- or non-cognate) tRNAs in order to
perform accurate decoding of a messenger RNA into proteins. The previous model of
decoding was based on crystal structures of the isolated 30S ribosomal subunit soaked with
short analogs of mRNA and tRNA. The analysis of our recent crystal structures of the full 70S
ribosome co-crystallized with its natural ligands suggested that the tRNA discrimination is
based on insisting on Watson-Crick geometry by a passive and rigid ribosomal decoding
center. We proposed that energy cost for formation of tautomers or repulsion energy within a
mismatch pair could constitute the sole cause for the very efficient rejection of near-cognate
tRNAs by the ribosome. Here we present new data that further describe the properties of the
ribosomal decoding center. We solved several high-resolution X-ray structures of the 70S
ribosome with near-cognate tRNAs that form different types of mismatches (pyrimidine-purine
or purine-purine) with the first two positions of the mRNA codon in the decoding center. We
also provide first X-ray data of the ribosome with a mismatch in the P site. This state is a result
of those rare events when a near-cognate tRNA is accommodated on the ribosome and
translocated to the P site. Overall, our data substantiate our model of decoding and suggest
that incorporation of a wrong amino acid into the polypeptide chain can be explained on the
basis of molecular mimicry.
Page 51
EMBO Conference Series: Protein Synthesis and Translational Control
16
GTP hydrolysis by EF-G synchronizes tRNA translocation on small and large
ribosomal subunits
Wolf Holtkamp1, Carlos E. Cunha1, Frank Peske1, Andrey L. Konevega2, Wolfgang
Wintermeyer1, Marina Rodnina1
1 MPI for Biophysical Chemistry, Germany
2 Petersburg Nuclear Physics Institute, Russian Federation
Presenter: Wolf Holtkamp
The translocation step of the protein elongation cycle entails a coordinated movement of two
tRNAs and the mRNA through the ribosome. The 3’ ends of the tRNAs can spontaneously
move towards their new destinations on the 50S subunit; however, the peptidyl transferase
reactivity of peptidyl-tRNA in the hybrid state remains low, indicating that the final
posttranslocation state is not reached. Translocation of the tRNA anticodons on the 30S
subunit is inherently very slow; it is catalyzed by elongation factor G (EF-G) and accompanied
by GTP hydrolysis. In this work, we use a dual-reporter fluorescence assay to monitor coupling
between GTP hydrolysis and full translocation on both ribosomal subunits in real time. We find
that EF-G-catalyzed translocation proceeds synchronously on 30S and 50S subunits, although
the movement on the two subunits can be uncoupled by antibiotics or mutations in EF-G.
Whereas rapid partial 50S translocation is induced by EF-G binding alone, 30S translocation as
well as the completion of 50S translocation requires GTP hydrolysis and the function of domain
4 of EF-G. These results reveal two distinct modes to utilize the energy of EF-G binding and
GTP hydrolysis, respectively, and suggest that the coupling between energy consumption and
tRNA-mRNA translocation is mediated through the 30S subunit. The ribosome–EF-G complex
appears to combine the features of a Brownian machine and a motor driven by a power-stroke
mechanism.
Page 52
Speaker Abstracts
17
Structure and structural dynamics of mammalian ribosomal complexes during
translation elongation
Tanya Budkevich1, Justus Loerke1, Elmar Behrmann1, David Ramrath1, Andrea Schmidt1,
Jochen Ismer1, Thorsten Mielke2, Knud Nierhaus3, Chang Shung4, Karissa Sanbonmatsu4,
Peter Hildebrand1, Patrick Scheerer1, Christian Spahn1
1 IMPB, Charité – Universitätsmedizin Berlin, Germany
2 Max-Planck Institut für Molekulare Genetik, UltraStrukturNetzwerk, Germany
3 Max-Planck Institut für Molekulare Genetik, Abteilung Vingron, Germany
4 Los Alamos National Laboratory, Germany
Presenter: Christian Spahn
The high degree of evolutionary and structural conservation in the core architecture of the
ribosome suggests that the basic mechanism of translation is similar in all domains of life.
Nevertheless, structural and functional differences of prokaryotic and eukaryotic ribosomes are
evident, among them size and composition, sensitivity to antibiotics, the increased
complication of the initiation process. Here we present cryo-EM maps of the mammalian 80S
ribosome in various functional states throughout the elongation cycle. The resolution for the
various maps ranges from sub-nanometer to near-atomic resolution. The POST complex is in
the classical conformation and contains two tRNAs in classical P/P and E/E positions. The
absence of the intersubunit rotation is a common feature for the actual POST complex and a
classical configured sub-population of the PRE complex. However, an unexpected kind of 40S
movement - rolling - is observed, when both maps are compared, which according to our
knowledge has not been observed so far in the bacterial system. Accordingly, a large-scale
conformational change has to occur in the mammalian system during the transition from the
POST to the PRE state, i.e. during decoding or tRNA accomodation. Interestingly, also the
mammalian decoding complex with aa-tRNA•eEF1A•GMPPNP exhibits differences to bacterial
one in terms of conformation and the interaction pattern between ternary complex and the
ribosome. Our finding emphasizes once again the distinctions in the complexity and/or
thermodynamic behavior between prokaryotic and eukaryotic ribosomes.
Page 53
EMBO Conference Series: Protein Synthesis and Translational Control
18
eIF5A promotes translation of Polyproline Motifs
Erik Gutierrez1, Byung-Sik Shin1, Christopher Woolstenhulme2, Joo-Ran Kim1, Preeti Saini1,
Allen Buskirk2, Thomas Dever1
1 NIH, United States of America
2 Brigham Young University, United States of America
Presenter: Thomas Dever
Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to
stimulate methionyl-puromycin synthesis, a model assay for peptide bond formation. More
recently, eIF5A was shown to promote translation elongation; however, its precise requirement
in protein synthesis has remained elusive. Using in vivo dual-luciferase reporter assays in yeast
and in vitro reconstituted translation assays we reveal a specific requirement for eIF5A to
promote peptide-bond formation between consecutive proline residues. Ribosomal
toe-printing assays reveal that addition of eIF5A relieves ribosomal stalling during translation of
three consecutive proline residues in vitro, and loss of eIF5A function impairs translation of
native polyproline-containing proteins in yeast cells. Hydroxyl radical probing experiments
localize eIF5A near the E site of the ribosome with its hypusine residue adjacent to the
acceptor stem of the P-site tRNA. Thus, we propose that eIF5A, like its bacterial ortholog EFP,
stimulates the peptidyl-transferase activity of the ribosome and facilitates the reactivity of poor
substrates like proline.
Page 54
Speaker Abstracts
19
A proline triplet in Val-tRNA synthetase explains the origin of EF-P and eIF5A
Agata Starosta1, Lauri Peil2, Gemma Aktinson3, Kai Virumäe4, Christopher Woolstenhulme5,
Allen Buskirk5, Tanel Tenson3, Jaanus Remme4, Daniel N. Wilson1
1 Gene Center Munich, LMU Munich, Germany
2 Wellcome Trust Centre for Cell Biology, University of Edinburgh, United Kingdom/ Institute
of Technology, University of Tartu, Estonia
3 Institute of Technology, University of Tartu, Estonia
4 Institute of Molecular and Cell Biology, University of Tartu, Estonia
5 Brigham Young University, United States of America
Presenter: Agata Starosta
During translation, amino acids are not incorporated into the growing nascent polypeptide
chain at the same rates. In fact, distinct amino acid sequences, such as polyproline stretches,
can cause translational arrest [1,2,3] which is alleviated by elongation factor P (EF-P) in
bacteria or eIF5A in eukaryotes [1,2,4]. Both, EF-P and eIF5A are post-translationally modified
by lysinylation and hypusination, respectively [5,6], and these modifications are crucial for
ability of the respective factors to alleviate ribosomal stalling [1,2,4]. It remains unclear why
evolution has favored the development of specialized factors, rather than simply selecting
against the occurrence of polyproline stretches in proteins - especially given that the activity of
EF-P and a/eIF-5A requires the co-evolution of specialized enzymes that post-translationally
modify these factors. We have discovered that only a single polyproline stretch is conserved
across all domains of life, namely, a proline triplet present in the valyl-tRNA synthetase (ValS).
Here we show that expression of ValS indeed requires the presence of modified EF-P.
Moreover, the proline triplet, which is located in the active site of ValS, is critical for efficient
charging of tRNAVal with valine, but also for preventing misactivation of non-cognate amino
acids such as isoleucine. Collectively, our findings indicate that the role of the essential proline
triplet for ValS activity has led to the evolution of the EF-P/IF5A rescue systems to overcome
the translation problems imparted by the presence of the proline triplet. Furthermore, our
results provide fundamental structural insight into the mechanism by which ValS discriminates
the cognate amino acid valine from the near-cognate isoleucine.
References:
[1] Ude et al. Science 2013; [2] Doerfel et al., Science 2013, [3] Woolstenhulme et al., PNAS
2013 [4] Gutierrez et al. Mol. Cell 2013; [5] Peil et al. NCB 2012, [6] Park et al. Amino acids
2010
Page 55
EMBO Conference Series: Protein Synthesis and Translational Control
20
Molecular dynamics simulations of eukaryotic ribosomes: integrating theory
and experiment
Serdal Kirmizialtin1, Suna Gulay2, Jonathan Dinman2, Scott Blanchard3, Marat Yusupov4,
1 New Mexico Consortium, United States of America
2 University of Maryland, United States of America
3 Cornell Medical College, United States of America
4 IGBMC, France
5 Charite, Germany
6 Los Alamos National Laboratory and New Mexico Consortium, United States of America
Presenter: Karissa Sanbonmatsu
We will report results from the first molecular simulations of eukaryotic ribosomes. Using an
integrated approach, we combine data from X-ray crystallography, cryo-EM and SHAPE
chemical probing. Over the past decade, we have developed a pipeline that begins with X-ray
crystallographic structures and uses molecular simulation to produce all-atom models
consistent with cryo-EM reconstructions. Theses models are then used as beginning and end
points for simulations of large-scale conformational changes. Our strategy has been highly
successful in the case of the accommodation conformational change during tRNA selection in
bacteria. Here, we correctly predicted the universally conserved accommodation corridor,
which has been verified in several independent experimental studies. We have also recently
identified the hybrid corridor, responsible for tRNA hybrid state formation during translocation.
Our latest addition to our pipeline is the incorporation of SHAPE probing data describing the
mobility of the RNA backbone in solution. We have developed a novel algorithm to generate
molecular dynamics simulations highly consistent with SHAPE probing data. We have applied
these techniques to eukaryotic ribosomes to investigate their dynamics and conformational
changes.
Page 56
Speaker Abstracts
21
The eEF2 kinase (eEF2K) promotes cell survival under acute nutrient
deprivation by blocking mRNA translation elongation
Poul H Sorensen1, Marc Remke2, Michael Pollak3, Russel G Jones3, Eric Jan4, Christopher
Proud5, Stefan Pfister6, Brent Derry2, Michael Taylor2, Gabriel Leprivier1
1 BC Cancer Research Centre, Canada
2 University of Toronto, Canada
3 McGill University, Canada
4 University of BC, Canada
5 University of Southampton, United Kingdom
6 German Cancer Research Center, Germany
Presenter: Poul H Sorensen
Nutrient deprivation is a prototypical form of stress that can severely compromise cell viability.
To protect cells when nutrients are scarce, energy-consuming processes such as proliferation
and protein synthesis must be attenuated. It is well documented that physiologic control of
mRNA translation at the initiation step is critical to prevent cell death under nutrient depletion.
We report that regulation of translation at the elongation step, through the action of the
eukaryotic translation elongation factor 2 kinase (eEF2K), is also critical for cellular protection
against acute nutrient deprivation. Indeed, eEF2K confers cell survival under acute nutrient loss
by phosphorylating eEF2 and directly inhibiting translation elongation. Tumor cells hijack this
pathway to support metabolic adaptation to nutrient deprivation through activation of an AMP
kinase (AMPK)-eEF2K axis by increasing their AMP/ATP ratios. In the absence of eEF2K, the
capacity of tumor cells to adapt to nutrient deprivation is severely impaired. In vivo, eEF2K
overexpression in tumor cells provides dramatic protection of tumors from caloric restriction.
The clinical relevance of these findings is highlighted by strong correlations between high
eEF2K expression and poor survival in the high-risk neural tumors medulloblastoma,
glioblastoma multiforme, and neuroblastoma. Moreover, the physiological response to nutrient
deprivation in C. elegans is severely compromised in worms deficient in efk-1, the eEF2K
ortholog. These data highlight a highly conserved role for eEF2K in protecting cells from
nutrient deprivation, and in conferring tumor cell adaptation to metabolic stress through
selective control of mRNA translation elongation. The consequences of eEF2K activation on
the landscape of global translation under nutrient deprivation are currently under investigation
using several proteomic approaches.
Page 57
EMBO Conference Series: Protein Synthesis and Translational Control
22
Efficient -2 frameshifting by mammalian ribosomes
Andrew Firth1, Ying Fang2, Emmely Treffers3, Yanhua Li2, Ian Brierley1, Sawsan Napthine1,
Susanne Bell1, Ali Tas3, Zhi Sun2, Yvonne van der Meer3, Arnoud de Ru3, Peter van Veelen3,
John Atkins4, Eric Snijder3
1 University of Cambridge, United Kingdom
2 South Dakota State University, United States of America
3 Leiden University Medical Center, The Netherlands
4 University of Utah/University College Cork, United States of America
Presenter: Andrew Firth
Programmed ‑ 1 ribosomal frameshifting (‑ 1 PRF) is used in the expression of many viral and
some cellular genes and the stimulatory elements have now been well-characterized. In
contrast, efficient natural utilization of ‑ 2 frameshifting was, until recently, unknown in
eukaryotic systems. Like all nidoviruses, members of the Arteriviridae (a family of positive-sense
single-stranded RNA viruses) express their replicase polyproteins pp1a and pp1ab from two
long ORFs (1a and 1b), where synthesis of pp1ab depends on ‑ 1 PRF. These polyproteins
are post-translationally cleaved into at least 13 functional nonstructural proteins (nsps).
Recently we found that porcine reproductive and respiratory syndrome virus (PRRSV), and
apparently most other arteriviruses, utilize a ‑ 2 frameshifting mechanism to express a
conserved ORF that overlaps the nsp2-encoding region of ORF1a in the ‑ 2/+1 frame. The
frameshifting occurs at a conserved G_GUU_UUU sequence (underscores separate ORF1a
codons) and is highly efficient (around 20% of ribosomes translating ORF1a make the ‑ 2
shift). Expression in PRRSV-infected cells of the product of ‑ 2 frameshifting, nsp2TF, was
verified using specific Abs, and the site and direction of frameshifting were confirmed via mass
spectrometric analysis of nsp2TF. Mutations preventing nsp2TF expression impair PRRSV
replication. While -1 PRF on a 'slippery heptanucleotide' sequence is normally stimulated by a
3'-proximal RNA stem-loop or pseudoknot structure, -2 PRF in the arteriviruses depends on a
strikingly different type of stimulator, with both a highly conserved CCCANCUCC motif 11 nt 3'
of the shift site and, uniquely, a virus-encoded trans-acting protein (nsp1beta) being essential
for efficient frameshifting. Our findings demonstrate for the first time natural utilization of ‑ 2
PRF in a eukaryotic system, and illustrate a radically new type of frameshift stimulator.
Page 58
Speaker Abstracts
23
Jmjd4 is a lysyl hydroxylase of eRF1 required for optimal translational
termination
Tianshu Feng, Atsushi Yamamoto, Pooja Singh, Roman Fischer, Luke Yates, Robert
Gilbert, Benedikt Kessler, Peter Ratcliffe, Mathew Coleman
University of Oxford, United Kingdom
Presenter: Mathew Coleman
The eukaryotic release factor, eRF1, binds to stop codons at the ribosomal A site to mediate
translational termination. We identified eRF1 as an interactor of Jmjd4, a member of an
oxygenase enzyme family that catalyse oxidative modifications. Mass spectrometry analysis of
endogenous human eRF1 revealed a +16 Da mass shift at K63, consistent with hydroxylation.
Subsequent analyses showed that K63 hydroxylation is near complete and ubiquitous across
multiple cell lines, tissues, and species. Jmjd4 activity was both necessary and sufficient for
this modification in vivo. Because K63 is part of a conserved functional motif within the stop
codon recognition domain, we postulated that Jmjd4-mediated hydroxylation of eRF1
promotes release factor activity. Indeed, we observed stop codon readthrough in multiple
contexts following Jmjd4 knockdown. We conclude that eRF1 K63 hydroxylation facilitates
release factor function, and in turn enables efficient translational termination. These results,
together with accounts of tRNA hydroxylation and our recent discovery of ribosomal protein
hydroxylation, indicate that protein translation is a major new target of oxygenase enzymes.
Page 59
EMBO Conference Series: Protein Synthesis and Translational Control
24
Regulated stop codon readthrough provides a mechanism for evolution of new
gene functions
Joshua Dunn1, Nicolette Belletir2, Elizabeth Gavis2, Weissman Jonathan3
1 University of California San Francisco, United States of America
2 Princeton University, United States of America
3 Department of Cellular and Molecular Pharmacology and Howard Hughes Medical
Institute, University of California San Francisco, United States of America
Presenter: Joshua Dunn
With remarkable fidelity, ribosomes terminate protein synthesis upon encountering a stop
codon. However, through both regulated mechanisms and errors, readthrough of stop codons
allows ribosomes to elongate rather than terminate the nascent peptide. Regulated stop codon
readthrough occurs in a diversity of viruses — where subpopulations of C-terminally extended
proteins are essential for reproduction — but has been observed in only a handful of eukaryotic
host genes. Intriguingly, phylogenetic studies have predicted readthrough to occur in a few
hundred genes in Drosophila melanogaster. However, the biological roles of readthrough
remain obscure due to a lack of experimental approaches to monitor it. Specifically, how
pervasive readthrough is in eukaryotes, the extent to which it is regulated by cellular
conditions, and the functional importance of the resulting protein extensions, remain elusive.
We have developed a ribosome profiling assay (deep sequencing of ribosome-protected
mRNA fragments) for Drosophila melanogaster. We uncovered nearly four hundred stop codon
readthrough events that yield C-terminally extended proteins. While our results support the
phylogenetic predictions, they indicate that readthrough is far more pervasive than expected:
the majority of extensions evolved recently within the melanogaster lineage and thus were not
predicted by phylogenetic approaches. Both the conserved and recently evolved extensions
are biologically significant: they bear signatures of protein-coding selection, contain functional
subcellular localization signals, and their synthesis is differentially regulated between cell types.
We further demonstrate that readthrough occurs in yeast and humans, indicating that
readthrough is a general feature of eukaryotic gene expression. By altering the C-termini of
proteins, stop codon readthrough provides a means for extant genes to acquire new functions,
adding plasticity to the proteome during both development and evolution.
Page 60
Speaker Abstracts
25
hiCLIP reveals the atlas of RNA structures recognized by Staufen 1
Yoichiro Sugimoto1, Jernej Ule1, 2
1
2
MRC Laboratory of Molecular Biology, United Kingdom
Department of Molecular Neuroscience, UCL Institute of Neurology, United Kingdom
Presenter: Jernej Ule
RNA structure plays an essential role in post-transcriptional regulation [1]. However, the nature
of RNA structures in vivo remains poorly understood, since existing methods cannot
interrogate inter-molecular and long-range RNA duplexes. To this end, we developed a highthroughput technique to directly identify RNA duplexes interacting with double-stranded RNA
binding proteins (dsRBPs) in an unbiased manner. This technique employs RNA hybridization,
UV cross-linking, immunoprecipitation and high-throughput sequencing (hiCLIP). We applied
hiCLIP to Staufen 1 (STAU1), a dsRBP that regulates mRNA stability and localization, and
thereby plays a crucial role in cellular differentiation and homeostasis [2]. We uncovered the
global characteristics of in vivo RNA structures bound by STAU1: the dominance of intramolecular RNA structures, the depletion of RNA structures from CDS of highly translated
mRNAs, and an unforeseen prevalence of long-range RNA structures in 3’ UTRs. We
demonstrated that STAU1 binds a long-range RNA structure in the 3’ UTR of X-box binding
protein 1 (XBP1) mRNA, and controls its cytoplasmic splicing during endoplasmic reticulum
(ER) stress. Moreover, we showed that STAU1 down-regulates mRNAs with STAU1-target
structures in their 3’ UTRs. Taken together, we reveal the characteristics and functions of RNA
structures that are bound by STAU1. The versatility of hiCLIP opens the way to understanding
the role of RNA structures in vivo.
References:
[1]
Wan, Y., Kertesz, M., Spitale, R.C., Segal, E. & Chang, H.Y. Understanding the
transcriptome through RNA structure. Nat Rev Genet 12, 641-655 (2011).
[2.]
Lebeau, G., et al. Staufen 2 regulates mGluR long-term depression and Map1b
mRNA distribution in hippocampal neurons. Learning & memory 18, 314-326 (2011).
Page 61
EMBO Conference Series: Protein Synthesis and Translational Control
26
A role for Puf3 in oxidative stress tolerance in S. cerevisiae
Chris Kershaw, Joe Costello, Lydia Castelli, William Rowe, Paul Sims, Simon Hubbard,
Chris Grant, Mark Ashe, Graham Pavitt
The University of Manchester, United Kingdom
Presenter: Chris Kershaw
Following stress, translation is reprogrammed to favour mRNAs that are required to respond to
the challenges posed by those conditions. The role of RNA binding proteins in altering an
mRNAs translational status is a critical point of control. Puf3 is a member of the Pumilio family
of RNA binding proteins and has been shown to play a role in the localisation of RNAs to
mitochondria as well as facilitating mRNA degradation. The in vivo targets and RNA specificity
of Puf3 have previously been investigated using RIP CHIP. This revealed several hundred
mRNA targets which share a common sequence in the 3’UTR. We used a RIP SEQ approach
to further investigate Puf3 targets. This approach yielded an increased number of transcripts
with the well-defined Puf3 binding site in the 3’UTR as well as other mitochondrial-targeted
transcripts. In addition transcripts containing a shorter Puf3 motif were also enriched,
indicating that our RIP SEQ approach provides a more comprehensive insight into
Puf3-associated mRNAs. puf3Δ transcriptome, translatome and proteome analyses provided
genome-wide profiles of the impact of Puf3 on the life of its bound mRNAs. We have also
observed a correlation between transcripts associated with Puf3 and transcripts which are
translationally repressed following oxidative stress. The functional consquence of this
association was reinforced by studies showing that a puf3Δ strain is more resistant to oxidative
stress, whilst many Puf3 target mRNAs were translationally up-regulated. Interestingly Puf3
itself associates with translating ribosomes and this association was lost following oxidative
stress. Thus we demonstrate that Puf3 plays a role in regulating protein synthesis of mRNAs
and is important for the oxidative stress response.
Page 62
Speaker Abstracts
27
CCR4 represses translation of meiotic genes in the fission yeast
Schizosacharomyces pombe
Alfredo Castello1, Bernd Fischer1, Sophia Foehr1, Anne-Marie Alleaume1, Tomaz Curk2,
Jeroen Krijgsveld1, Matthias Hentze1
1 EMBL Heidelberg, Germany
2 University of Ljubljana, Slovenia
Presenter: Alfredo Castello
The RNA interactomes of HeLa and HEK293 cells jointly comprise 1106 RNA-binding proteins
(RBPs) (1, 2), with almost half of these lacking well-defined RNA-binding domains (RBDs),
suggesting the existence of numerous unknown RNA-binding architectures. Here, we report
RBDmap, a new method built on interactome capture (3), to comprehensively identify the
RBDs of native RBPs in proliferative HeLa cells. Making use of in vivo UV-crosslinking of RBPs
to polyadenylated RNAs, capture on oligo(dT) magnetic beads, proteolytic mapping and mass
spectrometry combined with a sophisticated scoring algorithm, RBDmap “re-discovered” the
known RNA-binding sites (e.g. RRM, KH) of numerous well characterized RBPs, validating the
approach. Strikingly, RBDmap identified dozens of additional RNA-binding architectures (e.g.
thioredoxin, SSB, RAP, WD40) in multiple non-homologous proteins, also including disordered
motifs such as basic patches. RBDmap thus instructs on the modes of RNA-binding of
hundreds of proteins in their native cellular states, providing valuable structural and functional
insights into RNA biology. For example, the identification of the thioredoxin domain as a
high-confidence RBD reveals an intriguing link between the redox state of cells and RNA
metabolism.
References:
[1] A. G. Baltz et al., Mol Cell 46, 674 (2012). [2] A. Castello et al., Cell 149, 1393 (2012)
[3] A. Castello et al., Nat Protoc 8, 491 (2013).
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EMBO Conference Series: Protein Synthesis and Translational Control
28
Oncogenic properties of the RNA-binding protein UNR: Targets in melanoma
progression
Laurence Wurth1, Panagiotis Papasaikas1, María García2, Marisol Soengas2, Fátima
Gebauer1
1 Centre for Genomic Regulation, Spain
2 National Centre for Oncologic Investigations, Spain
Presenter: Laurence Wurth
Upstream of N-Ras (UNR/ CSDE1) is a conserved RNA binding protein involved in mRNA
regulation at the levels of translation and stability. In Drosophila, UNR binds to a large number
of transcripts, and some of these encode conserved factors which in mammals have been
related to cancer development [1]. We thus wondered whether mammalian UNR is involved in
cancer progression. Here we show that mammalian UNR has a strong oncogenic potential.
Using melanoma as a model, we have found that UNR is over-expressed in cancerous cells
compared to non tumoral melanocytes. Depletion of UNR from melanoma cells reduces
cancer-specific properties, such as anchorage-independent growth and clonogenicity.
Conversely, over-expression of UNR in human primary fibroblasts leads to cell transformation
in the absence of any other manipulation. In order to understand how UNR regulates
tumorigenicity at the molecular level, we identified direct targets of UNR using iCLIP. UNR
binds to ~ 8% of the melanoma cell transcriptome, including both coding and non-coding
genes. About 20% of the targets are also bound by UNR in Drosophila, suggesting conserved
UNR functions. A proportion of targets encode factors implicated in cancer development and
metastasis. According to a general role in post-transcriptional regulation, UNR binds with
strong preference to 3’ UTRs and coding sequences, while it is relatively depleted from introns
and 5’ UTRs. Thus, although mammalian UNR has been described as an ITAF (IRES
trans-acting factor), our data suggest that this role must be limited to a reduced number of
targets. In addition, only a small number of UNR targets show changes in follow-up
transcriptome analysis of control versus UNR-depleted melanoma cells. To gain insight into the
mechanisms used by UNR to regulate its targets, we are performing ribosome profiling
analysis. The results from these analyses will be discussed.
References:
[1] Mihailovich et al. 2012. RNA 18: 53-64.
Page 64
Speaker Abstracts
29
Understanding the translational regulation and oncogenic properties of
Musashi 1 by integration of Ribosome Profiling and target RNA
immunoprecipitation
Scott Kuersten
Epicentre (An Illumina Company), United States of America
Presenter: Scott Kuersten
The evolutionarily conserved RNA-binding protein Musashi1 (Msi1) is highly expressed in
glioblastoma, suggesting an oncogenic function. To characterize the potential role of Msi1 in
oncogenesis, we performed an extensive characterization of Msi using a variety of
genome-wide approaches including Ribosome Profiling, RIPseq and CLIP-seq and
miRNA-mediated regulation. These integrated techniques were designed to identify RNAs
targeted by Msi1 and their impact on the transcriptome and translational regulation. These
analyses reveal novel aspects of Msi1 mediated regulation that include a potential antagonism
with a specific set of miRNAs and further demonstrate that Msi1 modulates several cellular
aspects of tumorigenesis such as proliferation, apoptosis, “cancer stem cell survival”,
migration, and invasion. These observations are in agreement with the biological functions of
the mRNAs identified by both RIP and CLIP-type analysis as well as uncovering Msi1 targets of
translational control via ribosome profiling techniques. We propose that Msi1 is a viable
therapeutic target in glioblastoma by showing that inhibition of Msi1 expression using siRNAs
in a mouse xenograft model prevented tumor growth and identify the most relevant targets
involved in these processes. Our results suggest that Msi1 acts in an oncogenic manner and
promotes gliomagenesis through its post-transcriptional gene regulatory role.
Page 65
EMBO Conference Series: Protein Synthesis and Translational Control
30
Poly(A) RNA and Paip2 act as allosteric regulators of Poly(A)-binding protein
Seung Hwan Lee, Jungsic Oh, Jonghyun Park, Ki Young Paek, Sangchul Rho, Sung Key
Jang, Jong–Bong Lee Lee
Pohang University of Science & Technology (POSTECH), Republic of Korea
Presenter: Seung Hwan Lee
When bound to the 3¢ poly(A) tail of mRNA, poly(A)-binding protein (PABP) modulates mRNA
translation and stability through its association with various proteins. By visualizing individual
PABP molecules with poly(A) and/or PABP-interacting protein 2 (Paip2) in real time, we found
that the four disordered RNA recognition motifs (RRMs) that allow PABP to bind the poly(A) tail
adopt a conformation in which RRM1 is in close proximity to RRM4 due to the bending of the
region between RRM2 and RRM3 upon binding of PABP to poly(A). Paip2 actively disrupts the
bent structure of PABP by simultaneously interacting with RRM2 and RRM3, resulting in the
inhibition of PABP-poly(A) binding. These results suggest that the changes in the configuration
of PABP induced by interactions with various effector molecules, such as poly(A) and Paip2,
play pivotal roles in its function.
Page 66
Speaker Abstracts
31
Enzymes in posttranscriptional control: investigating the link between central
metabolism and gene regulation
Benedikt Beckmann1, Alfredo Castello1, Bernd Fischer1, Rastislav Horos1, Claudia Strein1,
Katrin Eichelbaum1, Sophia Föhr1, Thomas Preiss2, Lars Steinmetz1, Jeroen Krijgsveld1,
Matthias Hentze1
1 EMBL Heidelberg, Germany
2 The John Curtin School of Medical Research, Australia
Presenter: Benedikt Beckmann
We recently defined the mRNA interactome of proliferating human HeLa cells, identifying 860
mRNA-binding proteins (mRBPs) and implicating more than 300 previously unknown RBPs in
RNA biology [1]. Following the establishment of an adapted protocol for interactome capture,
we here report the next step forward, work on the unicellular yeast Saccharomyces cerevisiae.
Using quantitative proteomics, we identified 678 high confidence (m)RBPs (FDR<0.01),
including many previously unknown ones. Our data set includes 101 of the 120 recently
reported yeast RBPs [2], and additionally identifies 283 high confidence RBPs. Moreover,
integration of our data with published work on mammalian RBPs defines a first eukaryotic
„core interactome“ of 259 conserved RBPs. Strikingly, this core interactome includes several
RBPs involved in biochemical pathways, particularly of central carbon metabolism.
RNA-binding of these enzymes is poorly characterized to date; we have developed a novel
approach to identify the RNA-binding domains of RBPs and will discuss possible RNA-binding
sites of these enzymes as well as their conservation across eukaryotes emerging from our
data.
References:
[1] Castello A, Fischer B et al. Cell. 2012; 149(6) [2] Mitchell SF et al. Nat Struct Mol Biol. 2013;
20(1)
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EMBO Conference Series: Protein Synthesis and Translational Control
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Glutamyl-prolyl tRNA synthetase is a novel mTORC1-S6K1 effector that
negatively regulates translation of inflammation-related genes and determines
body mass
Paul Fox
Cleveland Clinic, United States of America
Presenter: Paul Fox
Glutamyl-prolyl tRNA synthetase (EPRS) is the unique component of the GAIT
(Interferon-Gamma Activated Inhibitor of Translation) complex that binds GAIT elements in the
3'UTR of inflammation-related mRNAs and inhibits their translation. Induced phosphorylation at
Ser886 and Ser999 in the non-catalytic linker domain of human EPRS orchestrates its release
from the parental multi-aminoacyl tRNA synthetase complex, assembly of the GAIT complex,
mRNA binding, and translational silencing activity. Cyclin-dependent kinase 5 (Cdk5), in
conjunction with regulatory protein Cdk5R1 (p35), induces the initial phosphorylation of EPRS
at Ser886. Now, we show that mammalian target of rapamycin complex (mTORC)1, in
coordination with Cdk5, phosphorylates ribosomal protein S6 kinase-1 (S6K1), which in turn
phosphorylates Ser999 to generate translation silencing-competent EPRS, establishing EPRS
as a novel mTORC1-S6K1 axis target. Remarkably, we observed that S6K1 exhibits an
unprecedented “kinase phospho-code” that determines target selectivity. mTORC1-dependent
S6K1 phosphorylation induces phosphorylation of RPS6, eIF4B, and eEF2K, all components of
the protein synthetic apparatus that stimulate translation globally. In contrast, dual S6K1
phosphorylation by mTORC1 and Cdk5 directs specific phosphorylation of EPRS that
selectively inhibits translation of an ensemble of inflammation-related genes. To investigate the
role of EPRS phosphorylation in vivo, we have generated a loss-of-function mouse strain with
knock-in of Ser999-to-Ala in the EPRS gene. Adult homozygous mutant mice exhibit markedly
reduced body weight and adipose tissue mass, approximately phenocopying S6K1-null and
adipocyte-specific mTORC1-null mice, implicating EPRS as a critical mTORC1-S6K1 effector
regulating metabolism and growth.
Page 68
Speaker Abstracts
33
DAZL regulates both the translation initiation and deadenylation of germ cell
mRNAs
Richard Smith1, Barbara Gorgoni2, Kelsey Grieve1, William Richardson1, Joel Smith3, Nicola
Gray1
1 University of Edinburgh, United Kingdom
2 University of Aberdeen, United Kingdom
3 MRC Human Genetics Unit, United Kingdom
Presenter: Nicola Gray
Germ cells store many mRNAs in a translationally silent manner, frequently with short poly(A)
tails. DAZL (DAZ-like) is a member of the D eleted in Az oopermia (DAZ) family of RNA-binding
proteins that have a critical and conserved role in germ cell development from man to worms.
We have previously shown that DAZL binds to the 3’ untranslated region (UTR) of a subset of
mRNAs that are essential for gametogenesis and activates their translation (1-3). Here, we
show that a direct protein-protein interaction between DAZL and PABP is sufficient to recruit
PABP to target mRNAs, even in the absence of a poly(A) tail, and that this interaction
stimulates initiation downstream of the initial cap-binding event. Moreover, we find that DAZL is
multifunctional, as it can also regulate the deadenylation of mRNAs to which it is bound. This
new activity offers an explanation for the changes in poly(A) tail length that can be observed
upon ectopic expression of DAZL in the soma of zebra-fish embryos (4) and the decreased
stability of some germ cell mRNAs in Dazl knock-out mice (3). Since changes in poly(A) tail
length are not required for the ability of DAZL to stimulate the translation of target mRNAs, we
present a model in which DAZL utilises PABP to activate the translation of mRNAs and then
maintains them in an actively translating state by protecting their poly(A) tails.
References:
[1] Collier et al (2005) EMBO J. 24: 2656-2666 [2] Reynolds (2007) RNA 13:974-981
[3] Reynolds (2005) Hum Mol Genet 14: 3899-3909. [4] Takeda et al (2009) PLoS One 4:
e7513 [5] Maratou et al (2004) Mol Reprod Dev. 67: 26-54
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EMBO Conference Series: Protein Synthesis and Translational Control
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Involvement of IF2 N domain in ribosomal subunit joining revealed from
architecture and function of the full-length initiation factor
Angelita Simonetti1, Stefano Marzi2, Isabelle M. L. Billas1, Albert Tsai3, Attilio Fabbretti4,
Alexander G. Myasnikov1, Pierre Roblin5, Andrea Vaiana6, Isabelle Hazemann1, Daniel
Eiler7, Thomas Steitz7, Joseph D. Puglisi3, Claudio O. Gualerzi4, Bruno Klaholz8
1 IGBMC, France
2 IBMC, France
3 Stanford University School of Medicine, United States of America
4 University of Camerino, Italy
5 SOLEIL synchrotron, France
6 Max-Planck-Institute for Biophysical Chemistry, Germany
7 Yale University, United States of America
8 Institute of Genetics and of Molecular and Cellular Biology, France
Presenter: Angelita Simonetti
Translation initiation factor IF2 promotes 30S initiation complex (IC) formation and 50S subunit
joining, which produces the 70S IC. The architecture of full-length IF2, determined by small
angle X-ray diffraction and cryo electron microscopy, reveals a more extended conformation of
IF2 in solution and on the ribosome than in the crystal. The N-terminal domain is only partially
visible in the 30S IC, but in the 70S IC it stabilizes interactions between IF2 and the L7/L12
stalk of the 50S and upon its deletion proper fMet-tRNAfMet positioning and efficient
transpeptidation are affected. Accordingly, fast kinetics and single-molecule fluorescence data
indicate that the N-terminus promotes 70S IC formation by stabilizing the productive sampling
of the 50S subunit during 30S IC joining. Together, our data highlight the dynamics of
IF2-dependent ribosomal subunit joining and the role played by the N-terminus of IF2 in this
process.
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Speaker Abstracts
35
Mechanism of the initiator tRNA binding to the ribosomal P-site in Escherichia
coli
Umesh Varshney, Laasya Samhita, Smriti Arora, Sunil Shetty
Indian Institute of Science, India
Presenter: Umesh Varshney
The accuracy of the initiator tRNA (tRNAfMet) selection in the ribosomal P-site is central to
initiation of protein synthesis. A highly conserved occurrence of three consecutive G-C base
pairs in the anticodon stem of tRNAfMet in all domains of life contributes to its preferential
selection in the P-site. How this feature is exploited by ribosomes has remained unclear. In E.
coli, initiator tRNAs are encoded by four genes (metZ, metW and metV at 63.5’ and metY at
71.5’). We isolated Escherichia coli strains, which allow initiation with the tRNAfMet mutant
lacking the three G-C base pairs. Molecular characterization of many such strains has shown
that diminished cellular abundance of the chromosomally encoded tRNAfMet allows efficient
initiation with the tRNAfMet mutants. Based on these observations, we have eliminated all
native initiator tRNA genes from the E. coli genome, and sustained the strain on
unconventional initiator tRNAs of mycoplasma origin. Some of these initiator tRNAs retain only
the middle G-C base pair. Further, we observed that under the initiator tRNA depleted
conditions, elongator tRNAs can also initiate suggesting a novel mechanism to generate
proteome diversity in the cell. Further, to determine the importance of the ribosomal elements
in initiator tRNA binding in the P-site, we mutated two of the methyltransferases, RsmD and
RsmB (which methylate residues 966 and 967, respectively in 16S rRNA), and/or the
C-terminal tail of the S9 ribosomal protein. The observations suggest that these ribosomal
elements have distinctive roles in monitoring the accuracy of the codon-anticodon pairing in
the P-site to select the correct reading frame in an mRNA.
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EMBO Conference Series: Protein Synthesis and Translational Control
36
Roles of eIF1 and 40S mRNA-exit channel protein Rps5e in the response to
start codon consensus sequence
Alan Hinnebusc1, Pilar Martin Marcos1, Jagpreet Nanda2, Jyothsna Visweswaraiah1, Yvette
Pittman1, Jon Lorsch2, Thomas Dever1
1National Institutes of Health, United States of America
2Johns Hopkins University School of Medicine, United States of America
Presenter: Alan Hinnebusch
In the model for initiation by scanning, eIF1 promotes an open 40S conformation competent
for loading the ternary complex (TC) in a conformation (“Pout”) capable of sampling triplets in the
P site, while blocking accommodation of Met-tRNAi in the “Pin” state and completion of GTP
hydrolysis (Pi release) by eIF2. These functions should be neutralized by eIF1 dissociation from
the preinitiation complex (PIC) on AUG recognition. We obtained unequivocal support for this
model by showing that eliminating direct eIF1-40S contacts weakens eIF1 binding to 40SeIF1A complexes in vitro and confers Gcd- and Sui- phenotypes indicating, respectively,
reduced TC loading to the open/Pout conformation and increased transition to the closed/Pin
state at UUG codons. Moreover, both phenotypes are suppressed by overexpressing the eIF1
mutants or by an eIF1A mutation (17-21) shown to delay eIF1 release from reconstituted PICs.
We also selected eIF1 Ssu- mutants that suppress the Gcd-/Sui- phenotypes conferred by loss
of an eIF1-40S contact, which stabilize 40S binding by eIF1 in vitro. The D61G Ssusubstitution in particular reduces the rate of eIF1 dissociation and destabilizes TC binding in
reconstituted PICs, confirming destabilization of the closed/Pin state. eIF1 also discriminates
against AUGs in poor sequence context and this activity is exploited to autoregulate translation
of its own (SUI1) mRNA. Interestingly, we found mutations in the beta-hairpin of 40S protein
Rps5e that exacerbate the effect of poor context at the SUI1 AUG, lowering eIF1 expression;
but they do not discriminate against UUG start codons after wild-type eIF1 levels are restored.
By contrast, mutations in the hairpin loop suppress UUG initiation but do not discriminate
against poor context at the SUI1 AUG. Thus, different portions of the Rps5e hairpin, which
extends into the mRNA exit channel near the context nucleotides, have distinct functions in
discriminating against poor context versus non-AUG codons.
Page 72
Speaker Abstracts
37
HCV-like IRESs sequester eIF3 to promote translation of their viral mRNA
Yaser Hashem1, Amedee des Georges1, Vidya Dhote2, Robert Langlois1, Robert A.
Grassucci1, Christopher Hellen2, Tatyana Pestova2, Joachim Frank1
1 HHMI-Columbia University, United States of America
2 SUNY Downstate Medical Center, United States of America
Presenter: Amedee des Georges
Initiation on Hepatitis C virus (HCV), Classical swine fever virus (CSFV) and other HCV-like
IRESs relies on their specific interaction with the 40S subunit, which places the initiation codon
directly into the P site, where it base-pairs with eIF2-bound Met-tRNAiMet, resulting in
formation of a 48S complex. All HCV-like IRESs also specifically interact with eIF3, but the role
of this interaction in IRES-mediated initiation remains unknown. However, comparison of the
ribosomal positions of eIF3 in the mammalian 43S complex (Hashem et al. 2013) and of the
HCV IRES (Spahn et al. 2001) revealed that they overlap, suggesting that their rearrangement
would be required to form a ribosomal complex containing them both. Here we present a
cryoEM reconstruction at a sub-nanometer resolution of a 40S ribosomal complex containing
both eIF3 and the CSFV IRES. Strikingly, compared to the 43S complex, eIF3 is completely
displaced from its ribosomal position and instead interacts exclusively with the apical region of
IRES domain III. Our results suggest that a role of the specific interaction of HCV-like IRESs
with eIF3 is to prevent ribosomal association of eIF3 by sequestering it, which would both
relieve the competition between the IRES and eIF3 for a common binding site on the 40S
subunit and reduce formation of 43S complexes, thereby favoring translation of viral mRNAs.
Page 73
EMBO Conference Series: Protein Synthesis and Translational Control
38
Histone H4 mRNA coding region contains a “Shine Dalgarno-like” sequence
allowing efficient translation initiation
Franck Martin1, Angelita SIMONETTI1, Laure Schaeffer1, Arnaud D'Orchymont1,
Jean-François Menetret2, Bruno Klaholz3, Gilbert Eriani1
1 IBMC - CNRS - UDS, France
2 IGBMC - CNRS - INSERM - UDS, France
3 Institute of Genetics and of Molecular and Cellular Biology, France
Presenter: Franck Martin
Replication-dependent histones are exclusively and massively produced during the S-phase of
the cell cycle. These proteins are expressed from a dedicated non-canonical subclass of
mRNAs which do not undergo splicing, contain unusually short UTRs and lack a polyA tail at
their 3’ extremity but instead end in a highly-conserved hairpin structure. Our laboratory has
shown that histone H4 mRNA translation is using a novel hybrid initiation mechanism that
combines canonical with IRES-like features. Indeed, the H4 mRNA coding region contains two
RNA elements that guide the tethering of the ribosome onto the AUG start codon in a
cap-dependent manner but without any scanning step (Martin et al., 2011). By using a novel
pull-down method, we purified H4 mRNA-programmed translation initiation complex from
rabbit reticulocyte lysates (Prongidi-Fix et al., 2013). Using cryo-EM we determined the 3D
structures of H4 mRNA-ribosome complexes. Additional density corresponding to H4 mRNA
at the entry of the mRNA channel reveals a direct interaction with 18S ribosomal RNA (rRNA).
This interaction drives the positioning of the AUG start codon in the P-site of the ribosome,
explaining the lack of scanning step. This is the first example of a eukaryotic ribosome
positioning by a direct interaction between a eukaryotic mRNA and the 18S rRNA. This new
type of translation initiation of histone H4 mRNA is reminiscent of prokaryotic translation
mechanism, which initiates translation through a direct interaction between 16S rRNA and the
upstream Shine Dalgarno sequence close to the mRNA exit site. In the case of H4 mRNA, the
interaction with the 18S rRNA occurs downstream of the AUG start codon at the mRNA entry
channel.
References:
Martin et al., Mol Cell (2011) 41, 197-209. Prongidi-Fix et al. Biochem J (2013) 449, 719-28.
Page 74
Speaker Abstracts
39
The tumor suppressor p53 acts as the safeguard of translational control
through direct regulation of fibrillarin expression and rRNA methylation in
cancer
Sandra Ghayad1, Virginie Marcel2, Stéphane Belin1, Anne-Pierre Morel1, Gabriel Thérizols1,
Eduardo Solano-Gonzàlez3, Julie Vendrell1, Sabine Hacot1, Hichem C Mertani4,
Marie-Alexandra Albaret1, Jean-Christophe Bourdon5, Lee Jordan5, Alistair Thompson1,
Yasmine Tafer1, Rong Cong6, Philippe Bouvet6, Jean-Christophe Saurin1, Frederic Catez2,
Anne-Catherine Prats3, Alain Puisieux1, Jean-Jacques Diaz1
1 CRCL, France
2 Cancer Research Center of Lyon, France
3 University of Toulouse, France
4 Cancer Research Center of Lyon, Inserm U1052 CNRS 5286 UCBL, France
5 University of Dundee, United Kingdom
6 ENS Lyon, France
Presenter: Virginie Marcel
Aberrant translational control is a critical feature in promoting cellular transformation. Alteration
of oncogenes and tumor suppressors have been shown to affect signaling pathways leading to
an overall increase in protein synthesis, but also in the translation of specific subsets of mRNA
that favor tumor progression. It also alters ribosome production, mainly resulting in an increase
in ribosome quantity in cancer cells. A growing body of evidence suggests that modulation of
ribosome composition can affect the intrinsic activity of ribosome in translational control. Within
ribosomes, translation relies on the ribozyme activity of ribosomal RNAs (rRNA), which are
highly chemically modified, including over 105 2'-O-ribose-methylation added by the
methyl-transferase fibrillarin. However, whether master genes regulate the intrinsic translational
activity of ribosomes is unknown. In this study, we show that the tumor suppressor p53 protein
directly down-regulated fibrillarin expression in several cellular models and in human breast
cancer samples. Consistently, p53 inactivation in cancer cells was associated with modification
of rRNA methylation pattern. Moreover bi-cistronic luciferase assays and analysis of
polysome-associated mRNA showed that p53 inactivation results in an increase in Internal
Ribosome Entry Site-dependent translational initiation of mRNAs of key cancer-related genes.
Finally, we found that high fibrillarin expression is an independent marker of poor breast cancer
prognosis. Our data show that p53 maintains translational integrity, thus acting as the
“safeguard of protein synthesis”, through the direct regulation of fibrillarin expression and the
subsequent regulation of ribosome quality and intrinsic activity. Deregulation of factors
controlling ribosome biogenesis, such as p53, could result in production of "pathological
ribosomes” that enhance the synthesis of a subset of proteins promoting cancer progression.
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EMBO Conference Series: Protein Synthesis and Translational Control
40
Translation acts upstream of transcription, in vivo
Daniela Brina1, Annarita Miluzio1, Sara Ricciardi1, Kim Clarke2, Jan Rozman3, Birgit
Rathkolb3, Susanne Neschen3, Eckhard Wolf3, Martin Klingenspor3, Valérie Gailus-Durner3,
Helmut Fuchs3, Martin Hrabê de Angelis3, Nina Offenhauser4, Francesco Falciani2, Stefano
Biffo1
1 San Raffaele Scientific Institute, Italy
2 University of Liverpool, United Kingdom
3 German Mouse Clinic, Germany
4 IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Italy
Presenter: Daniela Brina
Translation is a process linking nutrient and growth factor signalling to the execution of specific
metabolic programs. Metabolism plays an active role in determining the fate of the cell. It is not
known whether the activity of initiation factors can regulate metabolic activity. eIF6 regulates
60S availability in response to extracellular signals. A mouse model of eIF6 depletion displayed
reduced body weight and reduced liver and white adipose tissue weight, as well as cancer
resistance. In addition, heterozygote (het) hepatocytes showed a blunted insulin translational
response when compared to wild-type (wt) ones. This finding led us to investigate the
metabolic phenotype of eIF6 haploinsufficient mice. Here we show that reduced translation
downstream of insulin is associated with normal glucose tolerance and glucose uptake and,
paradoxically reduced blood insulin levels and glycemia, thus suggesting a metabolic
reprogramming. We analyzed the molecular grounds for this unexpected phenotype. In short,
we show that the intracellular glycolytic flux is reprogrammed favoring gluconeogenesis, and
that the lipid biosyntheythic pathway is inhibited. We demonstrate that specific transcriptional
changes, due to altered translation, underlies the metabolic switch. All these changes are
controlled by eIF6 activity in a cell autonomous fashion. In addition, we show that only
insulin-responsive tissues reprogram transcription downstream of eIF6 activity, and that
eIF6-regulated transcriptional signature is strikingly similar to the one obtained by HDAC
inhibitors. Further analysis identifies mRNAs regulated by eIF6 activity at the translation level. In
conclusion, the translation activity of eIF6 causes a metabolic reprogramming that involves
specific transcriptional changes in a cell autonomous fashion, suggesting that in vivo,
translation acts hierarchically upstream of transcription in regulating cellular metabolism.
Page 76
Speaker Abstracts
41
Introduction + Structural basis of termination and co-translational translocation
in eukaryotes
Roland Beckmann
Gene Center Munich, Germany
Presenter: Roland Beckmann
For translation termination the appearance of a stop-codon in the ribosomal A-site is
recognized in eukaryotes by the eRF1-eRF3 release factor complex. The decoding of the
stop-codon requires the so-called NIKS-motif in the N-terminal domain of eRF1. This event is
followed by GTP hydrolysis by eRF3 allowing eRF1 to catalyze the hydrolysis of the
peptidyl-tRNA involving a conserved GGQ motif in the central domain of eRF1. Notably,
termination can be stimulated by the ribosome-recycling factor ABCE1. Here, we present
cryo-EM structures of eRF1-eRF3 and eRF1-ABCE1 containing pre-termination and
termination/recycling complexes. The structures reveal a distinct order of factor interactions
and conformational changes that eventually trigger efficient peptidyl-tRNA hydrolysis. The
translocation of hydrophilic proteins and the membrane insertion of hydrophobic proteins can
be efficiently coupled to their synthesis by the ribosome. To that end the translating ribosome
binds directly to the protein-conducting channel, the Sec61 complex in eukaryotic cells. Here,
we present snapshots of the ribosome-Sec machinery engaged in co-translational membrane
translocation or insertion. In the presence of a transmembrane domain we observe a
conformation of the Sec complex with an opened lateral gate between helix 2 and 7. In
contrast, in the presence of a translocating hydrophilic peptide the Sec complex adopts a
closed conformation of the lateral gate.
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EMBO Conference Series: Protein Synthesis and Translational Control
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Structural basis of signal sequence surveillance and selection by the SRP-FtsY
complex
Ottilie von Loeffelholz1, Kèvin Knoops1, Aileen Ariosa2, Xin Zhang3, Manikandan
Karuppasamy1, Guy Schoehn4, Imre Berger1, Shu-ou Shan2, Christiane Schaffitzel1
1 EMBL Grenoble, France
2 California Institute of Technology, United States of America
3 The Scripps Research Institute, United States of America
4 Institut de Biologie Structurale Grenoble, France
Presenter: Christiane Schaffitzel
Signal-recognition particle (SRP)-dependent targeting of translating ribosomes to membranes
is a multistep quality-control process. Ribosomes that are translating weakly hydrophobic
signal sequences can be rejected from the targeting reaction even after they are bound to the
SRP. Here we show that the early complex, formed by Escherichia coli SRP and its receptor
FtsY with ribosomes translating the incorrect cargo EspP, is unstable and rearranges
inefficiently into subsequent conformational states, such that FtsY dissociation is favored over
successful targeting. The N-terminal extension of EspP is responsible for these defects in the
early targeting complex. The cryo-electron microscopy structure of this ‘false’ early complex
with EspP revealed an ordered M domain of Ffh (SRP protein) making two ribosomal contacts,
and the NG domains of Ffh and FtsY forming a distorted, flexible heterodimer. The less
favorable Ffh–FtsY NG domain arrangement observed in the ‘false’ early complex likely
impedes the formation of the stable closed complex, which is the subsequent step in the SRP
pathway and required to deliver the RNC to the membrane. Our structural data, underpinned
by quantitative thermodynamic and kinetic analyses, provide a rationale for the rejection of this
incorrect substrate from the SRP targeting pathway and a structural basis for SRP-mediated
signal-sequence selection during recruitment of the SRP receptor.
References:
Estrozi et al., Nat. Struct. Mol. Biol. (2011) 18, 88-90. Von Loeffelholz et al., Nat. Struct. Mol.
Biol. (2013) doi: 10.1038/nsmb.2546.
Page 78
Speaker Abstracts
43
Regulation of protein synthesis in bacterial dormancy by reversible
phosphorylation
Sandro Pereira, Jonathan Dworkin
Columbia University, United States of America
Presenter: Sandro Pereira
Many bacterial species can enter a reversible, non-growing dormant state in the presence of
environmental conditions that are not conducive for growth. The success of this survival
strategy is highly dependent on the tight regulation of both the entry into and exit from
dormancy. The spore-forming Bacillus subtilis is a bacterium with a well-defined dormant state.
During the onset of dormancy, B. subtilis expresses YabT, a eukaryotic-like Ser/Thr kinase,
which targets key components of the cellular translation machinery including Elongation factor
Tu (EF-Tu). We have found that YabT phosphorylates several EF-Tu residues including the
highly conserved Thr63 which lies within the GTPase active site of EF-Tu. Phosphorylation of
Thr63 significantly inhibits GTP hydrolysis that is necessary for the release of EF-Tu from the
ribosome following aa-tRNA delivery. We observed that phosphorylated EF-Tu is not released
from ribosomes both in vitro and in vivo and thereby acts to inhibit protein synthesis. Notably,
this inhibitory post-translational modification of EF-Tu can be reversed by the Ser/Thr
phosphatase PrpC. Thus, protein synthesis during dormancy is reversibly regulated by the
phosphorylation of EF-Tu on Thr63. YabT also phosphorylates other translational GTPases
including EF-G and IF2 on the equivalent Thr residue, suggesting that they are also subject to
this regulation. Since this Thr residue is conserved in all bacterial translational GTPases, we
expect this reversible phosphorylation to be a general mechanism underlying the regulation of
bacterial translation. Finally, eukaryotic mitochondria have a bacterial-like translation system
and the the Thr residue that is phosphorylated in bacterial EF-Tu63 residue is conserved in
mitochondrial EF-Tu, which suggests that this phosphorylation-dependent regulatory
mechanism is likely to be phylogenetically conserved.
Page 79
EMBO Conference Series: Protein Synthesis and Translational Control
44
Mechanisms for efficient tRNA translocation
Christian Blau1, Lars Bock1, Gunnar F. Schröder2, Iakov Davydov3, Niels Fischer1, Holger
Stark1, Marina Rodnina1, Andrea Vaiana1, Helmut Grubmuller1
1 MPI for Biophysical Chemistry, Germany
2 Forschungszentrum Jülich, Germany
3 SRC Bioclinicum, Russian Federation
Presenter: Christian Blau
During the elongation cycle, after peptide-bonds are formed in the ribosome, transfer RNAs
translocate to their new binding sites. Combining cryo-EM reconstructions of translocation
intermediates [1] with high resolution crystal structures, we obtained 13 near-atomic resolution
structures of the 70S ribosome during tRNA translocation and validated them using recent
crystal structures. From all-atom molecular dynamics simulations of these structures, we
estimated intrinsic transition rates between states for motions of the L1-stalk, the tRNAs and
intersubunit rotations. The obtained rate estimates suggest that tRNA movement, rather than
body and head rotation, is rate-limiting for most transitions between intermediate states of
tRNA translocation. By calculating the free energy of interaction between L1-stalk and tRNA,
we obtained molecular forces revealing that the L1-stalk is actively pulling the tRNA from P to E
site, and thereby overcomes barriers hindering tRNA motion. Further, ribosomal proteins L5
and L16 guide the tRNAs by 'sliding' and 'stepping' mechanisms involving conserved protein
residues, explaining how tRNA binding affinity is kept sufficiently constant to allow rapid
translocation despite large-scale displacements.
References:
[1] N. Fischer, A. Konevega, W. Wintermeyer, M. Rodnina and H. Stark, Nature, 2010, 466,
329-333
Page 80
Speaker Abstracts
45
Mechanistic insight into ribosomal -1 frameshifting in bacteria
Neva Caliskan, Frank Peske, Marina Rodnina
MPI for Biophysical Chemistry, Germany
Presenter: Neva Caliskan
Maintenance of the correct reading frame is one of the fundamental properties of the
ribosome. However, in the cases of programmed frameshifting the ribosome moves to an
alternative, overlapping reading frame upon encountering specific signals embedded in the
mRNAs. A variety of models has been proposed to explain frameshifting mechanistically,
resulting in a variety of different models. Importantly, it is unclear at which step of the
elongation cycle -1 frameshifting takes place. Here we examined -1 frameshifting by dissecting
individual steps of the elongation cycle using rapid kinetic methods. We employed a minimal
IBV 1a/1b frameshifting system, which leads to ~70% frameshifting in vivo measured by a dual
luciferase assay. We monitored amino acid incorporation at the slippery site as well as
following the slippery site. Our data show that frameshifting takes place following the unaltered
decoding of the slippery codons. At the codon following the slippery site, the pseudoknot
causes a translation pause during which the amino acids are incorporated significantly slower
at both -1 and 0 reading frames. The efficiency of -1 frameshifting does not depend on the
availability of aa-tRNAs at the A site. Rather, -1 frameshifting is a late translocation event,
which most likely takes place during mRNA and tRNA movement at the 30S subunit with EF-G
still bound to slowly translocating ribosomes. We also show that slippery sequence and
pseudoknot act in a concerted manner to stimulate -1 frameshifting. Together, these results
suggest the mechanism and exact timing of -1 frameshifting.
Page 81
EMBO Conference Series: Protein Synthesis and Translational Control
46
Small molecules on the eukaryotic ribosome
Nicolas Garreau de Loubresse, Irina Prokhorova, Gulnara Yusupova, Marat Yusupov
IGBMC, France
Presenter: Nicolas Garreau de Loubresse
Many aspects of translation and its regulation are specific to eukaryotes, whose ribosomes are
much larger and intricate than their bacterial counterparts. Recent advances in structure
determination of the yeast 80S ribosome at high-resolution revealed the precise architecture of
eukaryote-specific elements and their interaction with the universally conserved core. It
constitutes today an experimental framework to explore the eukaryotic translation apparatus as
well as small molecules of therapeutic interest for the treatment of infectious diseases, genetic
disorders and cancers. We report the first high-resolution crystal structures of several inhibitors
bound to the 80S ribosome.
Page 82
Speaker Abstracts
47
Peptidyl transferase inhibitors arrest ribosomes at specific amino acid codons
Sergey Dmitriev1, Kseniya Akulich2, Ivan Lomakin3, Pavel Sinitsyn2, Victoria Smirnova2,
Dmitry Andreev2, Ilya Terenin2, Andrey Mironov2, Ivan Shatsky2
1 Belozersky Institute of Physico-Chemical Biology, Russian Federation
2 Moscow State University, Russian Federation
3 Yale University, United States of America
Presenter: Sergey Dmitriev
Many antibiotics that target the eukaryotic ribosome represent potential antitumor agents. For
some of them, their molecular interactions have been extensively studied in structural terms
while functional details of their activity remain incompletely understood. Using toe-printing
technique, we analyzed effects of several translation elongation inhibitors in mammalian cell
extract. When pre-incubated with the lysate before mRNA addition, both aminoglycoside
antibiotics and translocation inhibitors stopped elongating ribosome at the very beginning of
the coding region. In contrast, several antibiotics that affected the peptidyl transferase center
(PTC) of the 60S subunit demonstrated complex patterns of toe-print signals along the mRNA.
We investigated this phenomenon in detail using a model of antitumor drug harringtonine,
which had been used recently in ribosome profiling studies. We showed that harringtonine and
its close derivative homoharringtonine specifically halted elongating ribosomes at Lys, Arg or
Tyr codons positioned in the P-site producing strong toe-print bands in the corresponding
positions. Bioinformatic analysis of ribosome profiling data generally confirmed this conclusion
in a transcriptome-wide scale. Molecular modeling based on recently resolved structures of
eukaryotic ribosome let us to assume that in addition to the spatial interference of
homoharringtonine in the A-site it may also require the interaction of the antibiotic with the
peptidyl group of the P-site bound tRNA. Finally, we unexpectedly revealed the same pattern
of elongation arrest for a chemically distinct trichothecene antibiotic, T-2 toxin, while another
PTC inhibitor of this group, diacetoxyscirpenol, produced a completely distinct pattern of
ribosome stops. Our data suggests that the mechanism of inhibition of the protein synthesis by
PTC inhibitors is more complex than it was originally proposed.
Page 83
EMBO Conference Series: Protein Synthesis and Translational Control
48
Allosteric control of the Ribosome by small-molecule Antibiotics
Michael Wasserman1, Leyi Wang1, Arto Pulk2, Roger Altman1, Jamie Cate2, Scott Blanchard1
1 Weill Cornell Medical College, United States of America
2 University of California, Berkeley, United States of America
Presenter: Scott Blanchard
Protein synthesis is targeted by numerous, chemically distinct antibiotics that bind and inhibit
key functional centers of the ribosome. Using single-molecule imaging and x-ray
crystallography, we have shown that the aminoglycoside neomycin blocks aminoacyl-transfer
RNA (aa-tRNA) selection, translocation and ribosome recycling by binding to helix 69 (H69) of
23S ribosomal RNA within the large subunit of the Escherichia coli ribosome. There, neomycin
prevents the remodeling of inter-subunit bridges normally accompanying the process of
subunit rotation to stabilize a partially rotated ribosome configuration in which P-site tRNA is
constrained in a previously unidentified hybrid position. Functional studies reveal that this
neomycin-stabilized intermediate is incompatible with translation factor binding.
Page 84
Speaker Abstracts
49
Gcn1 binding to the 40S ribosome head region is essential for fully activating
eIF2alpha kinase Gcn2.
Su Jung Lee1, Tanya Budkevich2, Andrew Cridge1, Viviane Jochmann1, Andrew Seberg3,
Mark Swanson4, Alan Hinnebusch5, Christian Spahn2, Evelyn Sattlegger1
1 Massey University, New Zealand
2 IMPB, Charité, Germany
3 University of Nebraska, United States of America
4 Mercer University School of Medicine, United States of America
5 NIH, United States of America
Presenter: Evelyn Sattlegger
In all eukaryotes, phosphorylation of eIF2α is a major mechanism to adjust protein synthesis to
stress. Amino acid deprivation leads to the accumulation of uncharged tRNAs, which in turn
causes the phosphorylation of eIF2α by the kinase Gcn2. It was first shown in Saccharomyces
cerevisiae that sensing amino acid shortages requires that Gcn2 binds directly to its effector
protein Gcn1, and both proteins must associate with translating ribosomes. Our hypothesis is
that Gcn1 is directly involved in transferring uncharged tRNAs from the ribosomal A-site to
Gcn2. Here, we provide evidence that Gcn1 contacts the small ribosomal protein RPS10. A
Gcn1 fragment encompassing amino acids 1060 to 1777 that is necessary for ribosome
binding, showed a yeast 2-hybrid interaction with RPS10A. Furthermore, the same Gcn1
fragment co-immunoprecipitated RPS10A. In vitro, purified Rps10A co-precipitated
Gcn1[1060 to 1777], and vice versa. RPS10B, which differs to RPS10A by only 3 amino acids,
also co-precipitated Gcn1[1060-1777]. Deletion of either RPS10A or RPS10B rendered cells
sensitive to amino acid analogues, suggesting that Gcn2 activation is impaired.
Overexpression of RPS10A and RPS10B, respectively, in otherwise wild-type strains also
rendered cells sensitive to amino acid analogues, and this sensitivity was exacerbated when
Gcn1 harbored a M7A mutation known to specifically weaken Gcn1-ribosome interaction.
RPS10 is located at the small ribosomal head region, and possible interactions of Gcn1 with
other RPSs in this region will be discussed. Our findings are consistent with our first results on
cryoEM analysis of the reconstituted Gcn1-ribosome complex. Together, our results support
the idea that contacting the small ribosomal head region is essential for Gcn1 function in
activating Gcn2 under amino acid starvation conditions.
Page 85
EMBO Conference Series: Protein Synthesis and Translational Control
50
Structure of the ribosome•SelB complex at near-atomic resolution
Niels Fischer1, Alena A. Paleskava1, Wang Zhe2, Gunnar F. Schröder2, Marina Rodnina1,
Holger Stark1
1 MPI for Biophysical Chemistry, Germany
2 Forschungszentrum Jülich, Germany
Presenter: Niels Fischer
The stop codon UGA normally signals the end of the ribosomal elongation phase. However, in
the presence of a specific hairpin structure in the mRNA, the so-called selenocysteine insertion
sequence (SECIS), the stop codon is recoded to the non-canonical amino acid selenocysteine
(Sec) by SelB, a specialized elongation factor. SelB recognizes the specific hairpin, while it
delivers Sec-tRNASec to the ribosomal A site. We have determined the structure of SelB in the
quarternary complex with SECIS RNA, Sec-tRNASec and GDPNP on the Escherichia Coli
ribosome at 4.2 Å resolution by electron cryomicroscopy (cryo-EM) using extensive
computational sorting. The cryo-EM structure allowed building a de novo backbone model for
bacterial SelB and tRNASec that reveals the architecture and unique features of the
SelB•GDPNP•Sec-tRNASec ternary complex. The EF-Tu like domains 1 to 3 of SelB bind to
the ribosome in a similar fashion as the EF-Tu ternary complex. Domain 4 of SelB, absent in
EF-Tu, extends to the mRNA entry tunnel on the 30S subunit. Additional structures of the
ribosome•SelB complex at sub-nanometer resolution show that winged-helix motif 4 of
domain 4 contacts the SECIS element at the mRNA entry tunnel in a dynamic fashion, whereas
winged-helix motif 2 is tightly bound to helix 16 of 16S rRNA. These additional contacts might
be important in modulating GTPase activation by SelB in comparison to the canonical
ribosome•EF-Tu complex. The present results provide a detailed snapshot view of the
ribosome upon recoding of the UGA codon with selenocysteine and illustrate the power of
cryo-EM to obtain near-atomic resolution structures of heterogeneous ribosome complexes
which are evasive targets for structure determination by X-ray crystallography.
Page 86
Speaker Abstracts
51
Structural insights into drug-induced ribosomal stalling
Stefan Arenz, Otto Berninghausen, Roland Beckmann, Daniel Wilson
University of Munich, Germany
Presenter: Stefan Arenz
Expression of the resistance-gene ermB depends on ribosome stalling during translation of the
upstream ermB leader peptide (ermBL). Thereby, the sequence of the nascent polypeptide as
well as the presence of an inducing drug (e.g. erythromycin) is crucial for
stalled-ribosome-complex (SRC) formation (reviewed by Ref. 1). Here we report a
cryo-electron-microscopy structure at 4-5 Å resolution providing the structural basis for our
model of how the stalling signal is communicated from the ribosome exit tunnel, via a
relay-mechanism, back to the peptidyl-transferase center (PTC) in order to inactivate the A-site
on the ribosome.
References:
[1] Vázquez-Laslop, N., Ramu, H., & Mankin, A. (2011). Nascent peptide-mediated ribosome
stalling promoted by antibiotics.
Page 87
EMBO Conference Series: Protein Synthesis and Translational Control
52
Mechanistic aspects of NMD in human cells
Oliver Mühlemann
University of Bern, Switzerland
Presenter: Oliver Mühlemann
Eukaryotic mRNAs with premature translation termination codons (PTCs) are recognized and
degraded through a process termed nonsense-mediated mRNA decay (NMD). To get more
insight into the recruitment of the central NMD factor UPF1 to target mRNAs, we mapped
transcriptome-wide UPF1-binding sites by individual-nucleotide-resolution UV cross-linking and
immunoprecipitation (iCLIP) in human cells and found that UPF1 preferentially associated with
3′ UTRs in translationally active cells but underwent significant redistribution toward coding
regions (CDS) upon translation inhibition. This indicates that UPF1 binds RNA before
translation and gets displaced from the CDS by translating ribosomes. Corroborated by RNA
immunoprecipitation and by UPF1 cross-linking to long noncoding RNAs, our evidence for
translation-independent UPF1-RNA interaction suggests that the triggering of NMD occurs
after UPF1 binding to mRNA, presumably through activation of RNA-bound UPF1 by aberrant
translation termination. Unlike in yeast, in mammalian cells NMD has been reported to be
restricted to cap-binding complex (CBC)–bound mRNAs during the pioneer round of
translation. However, we compared decay kinetics of two NMD reporter genes in mRNA
fractions bound to either CBC or the eukaryotic initiation factor 4E (eIF4E) in human cells and
show that NMD destabilizes eIF4E-bound transcripts as efficiently as those associated with
CBC. These results corroborate an emerging unified model for NMD substrate recognition,
according to which NMD can ensue at every aberrant translation termination event.
Page 88
Speaker Abstracts
53
How the interaction of PABPC1 with the translation initiation complex inhibits
nonsense-mediated decay of transcripts with an AUG-proximal nonsense
codon
Isabel Peixeiro, Alexandre Teixeira, Cristina Barbosa, Luísa Romão
Instituto Nacional de Saúde Dr. Ricardo Jorge, Portugal
Presenter: Luísa Romão
Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly
degrades mRNAs containing a premature termination codon (PTC). The unified model for NMD
proposes that the decision of NMD triggering is the outcome of the competition between the
cytoplasmatic poly(A)-binding protein 1 (PABPC1) and the NMD effector UPF1 for the
termination complex. Consequently, PTCs located far, in a linear sense, from the poly(A) tail
and associated PABPC1, in mRNAs containing downstream exon junction complexes (EJCs),
are expected to elicit NMD. Nevertheless, we have reported that human b-globin mRNAs
containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can
substantially evade NMD. We have reported that translation termination at an AUG-proximal
PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. In fact, we have
shown that the establishment of an efficient translation termination reaction at the
AUG-proximal PTC is dependent on PABPC1 interaction with the initiation factor eIF4G and
with the release factor eRF3 at the terminating ribosome. These interactions underlie critical
3’-5’ linkage of translation initiation with efficient termination at the AUG-proximal PTC and
contribute to an NMD-resistant PTC definition at an early phase of translation elongation.
Furthermore, we provide strong evidence that the eIF3 is involved in delivering
eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. This work corroborates
a role for PABPC1 on NMD evasion of transcripts carrying an AUG-proximal PTC and provides
further insights into the mechanistic details of PTC definition and translation initiation.
Page 89
EMBO Conference Series: Protein Synthesis and Translational Control
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The RQC complex in constant motion: from stalled 60S recognition to aberrant
nascent peptides degradation
Quentin Defenouillère, John Mouaikel, Abdelkader Namane, Cosmin Saveanu, Alain
Jacquier, Micheline Fromont-Racine
Institut Pasteur, France
Presenter: Quentin Defenouillère
Ribosome stalling on eukaryotic mRNAs triggers cotranslational RNA and protein degradation
through conserved mechanisms. In particular, messenger RNAs lacking a stop codon are
degraded by the exosome in association with the SKI complex, whereas the corresponding
aberrant nascent polypeptides are ubiquitinated by the E3 ligase Ltn1 and thus targeted to the
proteasome for degradation. However, the steps between translational arrest and polypeptide
degradation remain unclear. Genetic screens using SKI and LTN1 mutants allowed us to
identify two other partners, translation associated element 2 (Tae2) and ribosome quality
control 1 (Rqc1), which where physically associated to 60S ribosomal subunits with Ltn1, the
AAA-ATPase Cdc48, and its co-factors Ufd1-Npl4. Tae2, Rqc1 and Cdc48 were all required
for the degradation of polypeptides synthesized from Non-Stop mRNAs (Non-Stop protein
decay). Both Ltn1 and Rqc1 were essential for the recruitment of Cdc48 to 60S particles.
Polysome gradient analyses of mutant strains revealed unique intermediates of this pathway,
showing that the polyubiquitination of Non-Stop peptides is a progressive process. We
propose that ubiquitination of the nascent peptide starts on the 80S and continues on the 60S,
on which Cdc48 is recruited to extract the substrate from the 60S exit tunnel and to escort it
for proteasomal degradation.
References:
Defenouillère Q. et al, PNAS, 2013, 110(13):5046-51. Cdc48-associated complex bound to
60S particles is required for the clearance of aberrant translation products.
Page 90
Speaker Abstracts
55
Assembly and function of the CCR4-NOT complex
Andreas Boland, Ying Chen, Tobias Raisch, Stefanie Jonas, Duygu Kuzuoglu-Öztürk, Lara
Wohlbold, Oliver Weichenrieder, Eric Huntzinger, Elisa Izaurralde
Max Planck Institute for Developmental Biology, Germany
Presenter: Elisa Izaurralde
The CCR4-NOT complex plays a crucial role in post-transcriptional mRNA regulation in
eukaryotic cells. It catalyzes the removal of mRNA poly(A) tails, thereby repressing translation
and committing mRNAs to degradation. The complex consists of a catalytic module
comprising two deadenylases (POP2/CAF1 and CCR4) and the NOT module minimally
containing the NOT1, NOT2 and NOT3 subunits. Additional subunits within the complex have
been described, including NOT4, CAF40 and the species-specific subunits CAF130, NOT10
and NOT11. NOT1 functions as a modular scaffold to provide binding sites for the
NOT10-NOT11 complex at its N-terminus, the CAF1-CCR4 catalytic module and CAF40 in its
middle region, and the NOT2 and NOT3 subunits at the C-terminus. Thus, NOT1 is essential
for the assembly of the complete CCR4-NOT complex. With the exception of the catalytic
module, the molecular details underlying the assembly of the different modules and how they
dock onto the NOT1 scaffold remain largely unknown. To provide the missing structural
framework for understanding the assembly and functions of the CCR4-NOT complex, we
previously determined the crystal structure of human CAF1 in complex with a central MIF4G
domain in NOT1. Here we report the crystal structures of a ternary complex formed by the
C-terminal domains of human NOT1, NOT2 and NOT3 as well as of a binary complex formed
by CAF40 and the middle region of NOT1. Through a combined mutational and functional
analysis, we have identified critical residues required for deadenylation in vivo. Collectively, our
data provide a framework for understanding the role of the CCR4-NOT complex in
post-transcriptional mRNA regulation and provides the foundation for studies on the
mechanism of action of translational regulators such as the Nanos, Bicaudal C, Smaug and
GW182 proteins, which recruit the CCR4-NOT complex to their targets.
Page 91
EMBO Conference Series: Protein Synthesis and Translational Control
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The structural basis for the activity of a cytoplasmic RNA uridylyltransferase
Luke Yates1, Sophie Fleurdépine2, Chris Norbury2, Robert Gilbert1
1 University of Oxford, United Kingdom
2 Sir William Dunn School of Pathology, University of Oxford, United Kingdom
Presenter: Luke Yates
MicroRNAs (miRNAs) are versatile regulators of gene expression in higher eukaryotes. For
precise silencing of many different target mRNAs, miRNA stability and efficacy is controlled by
highly developed regulatory pathways and fine-tuning mechanisms both affecting miRNA
biogenesis and altering mature miRNA target specificity. In metazoans, the remodelling of the
3' ends of precursor miRNA (pre-miRNA) during their biosynthesis, by the cytoplasmic terminal
uridylyltransferases (TUTs) ZCCHC6 and ZCCHC11, can both protect from nucleases and
promote maturation (mono-uridylylation) or inhibit Dicer processing and promote decay
(oligo-uridylylation). We present X-ray crystal structures of the Schizosaccharomyces pombe
cytoplasmic RNA terminal uridylyltransferase, Cid1, the orthologue of the mammalian ZCCHC
enzymes. The structure of Cid1 in complex with UTP reveals that its nucleotide selectivity is
mediated by a single histidine residue that is conserved in the catalytically active
nucleotidyltransferase domains of ZCCHC6/ZCCHC11. In yeast, Cid1 binds to and uridylates
mRNAs to target them for degradation, but lacks a canonical RNA recognition motif. We found
that Cid1 interacts with RNA with high-affinity that is mediated by three basic surface patches
of amino acid side chains, which are non-contiguous in the primary sequence, and forms a
basic stripe across the surface that is essential for enzymatic activity. The homologous
mammalian nucleotidyltransferases ZCCHC6 and ZCCHC11 are also functionally similar to
Cid1 and their inhibition in Lin28A-expressing human cancer cells results in suppression of
invasiveness and tumorigenicity. In addition, we have begun to investigate the structural and
mechanistic basis for the activity of the ZCCHC6/11 enzymes.
Page 92
Speaker Abstracts
57
Structural and functional analysis of the spliceosomal RNP remodeling
enzyme, Brr2
Karine Santos1, Sina Mozaffari-Jovin2, Gert Weber1, Reinhard Lührmann2, Markus Wahl1
1 Free University Berlin, Germany
2 Max-Planck-Institute of Biophysical Chemistry, Germany
Presenter: Karine Santos
Splicing entails the removal of introns from eukaryotic pre-mRNA and the ligation of the
neighboring exons and is carried out by the spliceosome. An active spliceosome evolves on
the substrate by a stepwise assembly driven by members of the SF2 helicases. The Brr2
helicase is the key player in the catalytic activation process. Brr2 is exceptionally large and
contains two expanded helicase units fused in tandem. The crystal structure of a 200 kDa
portion of Brr2 showed that its two helicase units intimately interact with each other. The
inactive C-terminal unit strongly stimulates the N-terminal helicase. Using structure-guided
mutagenesis and pre-steady state kinetics, we delineated communication lines between the
cassettes and probed nucleotide binding preferences for each cassette. Our results revealed
the structural and functional interplay between two helicase cassettes in an SF2 enzyme and
suggested how Brr2 interactions may achieve regulation of the N-terminal helicase.
Page 93
EMBO Conference Series: Protein Synthesis and Translational Control
58
Using Ribosome footprinting to understand translational regulation during
vertebrate development
Antonio Giraldez
Yale University, United States of America
Presenter: Antonio Giraldez
To be announced
Page 94
Speaker Abstracts
59
Designing a stress resistant translation machinery - lessons from
trypanosomatids
Alexandra Zinoviev1, Melissa Leger2, Gerhard Wagner2, Michal Shapira1
1 Ben-Gurion University of the Negev, Israel
2 Harvard Medical School, United States of America
Presenter: Michal Shapira
Extreme temperatures cause a global translation arrest, preventing the accumulation of
polypeptides that risk misfolding. This is common in digenetic parasites that migrate between
invertebrates and mammals. Since temperature and pH switches drive the developmental
program of gene expression in trypanosomatids, both canonical and alternative pathways for
translation are required. Under conditions that mimic transmission to the host, the canonical
translation initiation complex (LIF4E-4) disintegrates, and an alternative cap-binding complex
comes into play (LIF4E-1), pulling down a multitude of initiation factors, except for any eIF4G
ortholog. We have obtained crystals of LIF4E-1, in attempt to solve its unusual mode of
regulation. Trypanosomatids do not express any homolog of 4E-BP, but a novel and
non-conserved 4E-Interacting protein (L4E-IP, 85 kDa) regulates the binding of LIF4E-1. A
nutritional stress experienced within the fly is known to induce parasite virulence, and prepare
for stage differentiation. Another paralog, LIF4E-3, has been assigned a function during
starvation. A mutation in the cap-binding pocket impairs its cap-binding activity, preventing its
ability to compete with the canonical factors. Instead, it enters into granules that are induced
during starvation, most probably to accompany and protect inactive RNAs. LIF4E-3 is
regulated by a novel eIF4G-like protein that under normal conditions sequesters it in the
cytoplasm, and releases it during nutritional stress to enter specific stress granules that are
devoid of DHH1. We highlight how orthologs of conserved factors have been recruited during
evolution to acquire novel functions that promote survival under harsh conditions.
Page 95
EMBO Conference Series: Protein Synthesis and Translational Control
60
A rice Cis-natural antisense RNA enhances PHO1;2 protein level via
Translational control and contributes to phosphate homeostasis and plant
fitness
Mehdi Jabnoune1, David Secco1, Cécile Lecampion2, Christophe Robaglia2, Qingyao Shu3,
Yves Poirier1
1 University of Lausanne, Switzerland
2 University Aix Marseille, France
3 University of Zhejiang, China
Presenter: Yves Poirier
Cis-natural antisense transcripts (cis-NATs) are widespread in eukaryotes and most often
associated with down-regulation of their associated sense genes. Unexpectedly, we found that
a cis-NAT positively regulates the level of a protein critical for phosphate homeostasis in rice.
OsPHO1;2, a gene involved in phosphate loading into the xylem, and its associated cis-NAT
(cis-NATPHO1;2) are both controlled from promoters active in the same cells in the vascular
cylinder. Under phosphate deficiency, expression of both cis-NATPHO1;2 and the OsPHO1;2
protein increased while the sense OsPHO1;2 mRNA level remained stable. Down-regulation of
cis-NATPHO1;2 expression by RNA interference resulted in a decrease in OsPHO1;2 protein,
impaired the transfer of phosphate from root to shoot and decreased seed yield. Constitutive
over-expression of NATPHO1;2 in trans led to a strong increase of OsPHO1;2 protein, even
under phosphate-sufficient conditions. Under all conditions tested, no changes occurred in the
steady-state level of the sense OsPHO1;2 mRNA and the splicing pattern remained
unchanged. Furthermore, no polypeptides originating from potential short open reading frames
present in cis-NATPHO1;2 were detected, indicating that it is most likely non-coding.
Modulation of cis-NATPHO1;2 expression was not associated with changes in the nuclear
export of the sense OsPHO1.2 mRNA, nor was it associated with editing of the mRNA.
However, expression of cis-NATPHO1;2 was associated with an enhanced association of both
OsPHO1;2 mRNA and the cis-NATPHO1;2 with polysomes. Together, these data indicate an
unexpected role for a cis-NAT in promoting translation of its cognate mRNA and provide a
novel link between antisense RNA and phosphate homeostasis.
Page 96
Speaker Abstracts
61
tiRNAs assemble G-quadruplex structures to inhibit translation initiation
Pavel Ivanov1, Elizabeth O'Day2, Mohamed Emara1, Gerhard Wagner2, Judy Lieberman2,
Paul Anderson1
1 Brigham and Women's Hospital/Harvard Medical School, United States of America
2 Harvard Medical School, United States of America
Presenter: Pavel Ivanov
Cleavage of tRNA is an evolutionary conserved phenomenon serving different functions in
different organisms. Angiogenin is a stress-induced ribonuclease that cleaves tRNA in the
ancicodon loop to produce tRNA-derived stress-induced RNAs (tiRNAs). We have previously
showed that selected tiRNAs inhibit protein synthesis in phospho-eIF2alpha-independent
manner to target translation initiation. We identified a terminal oligoguanine (TOG) motif of the
selected tiRNAs to be required for translation inhibition and stress response. Further
investigations reveal that TOG-possesing tiRNAs assemble unique G-quadruplex structures.
We also identified YB-1 protein as a direct interaction partner of tiRNAs that contributes to
angiogenin-, tiRNA- and oxidative stress-induced translation inhibition coupled to the assembly
of Stress Granules (SGs). We show that both RNA-binding domains (RBDs) of this protein, the
cold-shock domain (CSD) and the oligomerization domain, are indispensable for binding to the
G-quadruplex structures of tiRNAs. Interestingly, DNA analogs of tiRNAs (tiDNAs), which
structurally mimic tiRNAs, also interact with YB-1 and inhibit translation initiation, trigger
formation of SGs and induce stress response. Moreover, tiDNAs can be spontaneously
up-taken by cells and confer cytoprotection against variety of stresses. We believe that these
properties of tiDNAs are of potential therapeutic use for the treatment of neurodegenerative
diseases.
Page 97
EMBO Conference Series: Protein Synthesis and Translational Control
62
miR-17-92 controls MYC-centered regulatory networks to sustain growth of
MYC-dependent lymphomas
Marija Mihailovic1, Michael Bremang1, Elena Vitale1, Gabriele Varano2, Francesco Mancuso3,
David Cairns4, Stefano Casola2, Tiziana Bonaldi1
1 European Institute of Oncology (IEO), Italy
2 IFOM Foundation, the FIRC Institute of Molecular Oncology Foundation, Italy
3 Center for Genomic Regulation (CRG), Spain
4 Leeds Cancer Research UK Centre, United Kingdom
Presenter: Marija Mihailovic
The functional effect of miRNA activity depends on its molecular environment, having the
potential to play different roles in cancer development and maintenance based on changes in
the gene expression landscape. Although the role of miR-17-19b, a truncated version of the
miR-17-92 cluster, has been extensively studied during tumorigenesis, its role in the
maintenance phase of the tumor remains elusive. Here we dissected the regulatory network of
miR-17-19b in an established MYC-dependent B-cell lymphoma. Surprisingly, a mild
overexpression of the cluster interfered with the growth of this highly aggressive
MYC-dependent lymphoma both in vivo and in vitro, as a consequence of decreased
proliferation and increased apoptosis. SILAC-based quantitative proteomics led to the
identification of over 200 novel miR-17-19b targets, 40% of which were under the
transcriptional control of MYC, indicating a widespread silencing of MYC-centered regulatory
networks. Interestingly, even without being a direct miR-17-19b target, MYC itself was found
to be down-regulated. Polysomal analysis revealed that myc -mRNA is less efficiently
translated when miR-17-19b was increased. It was previously shown that RNA-binding protein
HuR modulates the stability and translation of myc -mRNA and that the association of HuR
with myc -mRNA was regulated by Chek-2 dependent HuR phosphorylation. Our global
proteomics analysis has uncovered Chek-2 as a novel miR-17-19b target, pointing towards
the possible involvement of the Chek-2/HuR axis in the regulation of MYC. We are currently
investigating the contribution of the Chek-2/HuR axis in the down-regulation of MYC and the
relevance of this down-regulation to the less aggressive phenotype observed upon
miR-17-19b overexpression. In conclusion, we propose that miR-17-19b supports tumor
maintenance by protecting the cell from the harmful effects caused by high MYC levels,
ensuring a fine control of the MYC level and function.
Page 98
Speaker Abstracts
63
Interplay between microRNA-21 and RNA-binding protein HuR in translation
regulation of the pro-inflammatory tumor suppressor gene Programmed Cell
Death 4 (PDCD4)
Dipak Poria1, Ipsita Nandi1, Partho Sarothi Ray1
1 Indian Institute of Science Education and Research, Kolkata, India
Presenter: Partho Sarothi Ray
Translation control of gene expression is mediated by the signal-dependent binding of
microRNAs and regulatory proteins to mRNAs. We have tried to systematically understand the
integration of these two modes of control in the regulation of inflammatory gene expression.
Translation regulation of pro-inflammatory genes plays an important role in the inflammatory
response. Disruption of such regulatory mechanisms leads to chronic inflammatory conditions,
including cancer. The pro-inflammatory tumor suppressor protein PDCD4 plays an important
role in maintaining the balance between inflammation and tumorigenesis. miR-21, an
oncogenic miRNA upregulated in many cancers, inhibits the translation of PDCD4 mRNA by
binding to its 3'UTR. We found that AU-rich element-binding protein HuR binds to the PDCD4
3'UTR and overexpression of HuR in human breast carcinoma cells inhibited miR-21-mediated
repression of PDCD4 translation. The tumor extracellular matrix component hyaluronan (HA)
represses PDCD4 expression by upregulating miR-21; however exposure of the HA-treated
cells to UV irradiation enhanced HuR level and restored PDCD4 expression. A cell line stably
expressing miR-21 showed higher rate of proliferation, which was reduced by HuR expression.
Therefore HuR could prevent miR-21-mediated repression of PDCD4 and consequent
oncogenic cell behavior. We delineated the HuR-binding sites in the PDCD4 3'UTR and HuR
was found to bind to two independent sites in proximity to the miR-21 target site. Remarkably,
removal of both sites still allowed HuR-mediated rescue of PDCD4 translation repression by
miR-21, suggesting that HuR might also be acting in trans. We unprecedentedly found that
HuR binds directly to miR-21, thereby preventing its interaction with the PDCD4 3'UTR. This
opens up the possibility of regulation of miRNA activity by direct binding to hitherto known
mRNA-binding proteins, resulting in fine-tuned gene expression in complex regulatory
environments.
Page 99
EMBO Conference Series: Protein Synthesis and Translational Control
64
RAN translation across intronic DM2 (CCTG) and ALS/FTD (GGGGCC)
expansion mutations
Laura Ranum1, Yuanjing Liu1, John Cleary1, Tammy Reid1, Monica Banez Coronel1, Juan
Troncoso2, Tao Zu1
1 University of Florida, United States of America
2 Johns Hopkins University, United States of America
Presenter: Laura Ranum
For a group of neurological diseases caused by microsatellite expansions, mutations within
predicted coding or non-coding regions are thought to cause disease by protein or RNA
mechanisms. In 2011, we discovered that in the absence of an AUG initiation codon,
expanded CAG repeats can express homopolymeric proteins from all three reading frames.
We showed this repeat-associated non-ATG (RAN) translation is hairpin-dependent, occurs
without RNA editing and is observed in cell culture as well as spinocerebellar ataxia type 8
(SCA8) and myotonic dystrophy type 1 (DM1) tissues. We now provide evidence that RAN
translation is a general mechanism that occurs across a variety of disease-causing expansion
motifs, including the CCTG tetranucleotide- and GGGGCC hexanucleotide-expansion
mutations which cause myotonic dystrophy type 2 (DM2) and amyotrophic lateral sclerosis /
frontotemporal dementia (C9ORF72 ALS/FTD), respectively. These intronic mutations have
been previously shown to form nuclear RNA foci. We now demonstrate that CCUG and
GGGGCC expansion transcripts accumulate in both the nucleus and cytoplasm in patient
tissues. The CCTG expansion mutation produces a tetra-repeat expansion protein with a
Leu-Pro-Ala-Cys expansion in each of three reading frames. The GGGGCC expansion
mutation produces dipeptide expansion proteins with Gly-Ala, Gly-Pro or Gly-Arg expansion
motifs. Cell culture studies show RAN translation of these repeats occurs with as few as 30
repeats. We have generated antibodies to detect novel RAN proteins in vivo and show
ALS/FTD-dipeptide and DM2-tetrapeptide proteins accumulate as protein aggregates in
patient autopsy brains. These results have implications for understanding fundamental
mechanisms of protein synthesis and translational control and should now be considered for a
broad category of neurological disorders.
Page 100
Speaker Abstracts
65
System wide analyses have underestimated transcriptional importance in
animals
Jingyi Jessica Li1, Peter Bickel1, Mark Biggin2
1 UC Berkeley, United States of America
2 Lawrence Berekely Laboratory, United States of America
Presenter: Mark Biggin
Historically transcription was viewed as the dominant step determining protein expression
levels. More recently system wide analyses have suggested that translation and other post
transcriptional steps play the dominant role, suggesting for example that differences in mRNA
expression between genes explain only 10-40% of the differences in protein levels. We have
critically reexamined these recent claims. We find that the mass spectrometry data used to
measure protein abundances has been inaccurately scaled non-linearly and underestimates
protein abundances ~ten fold compared to individual measurements for 61 housekeeping
proteins. In addition, we show that molecule-specific stochastic and systematic measurement
errors in mass spectrometry and mRNA-Seq data contribute significantly to the poor
correlation between measured mRNA and protein expression levels. We also show that the
system wide studies have underestimated the importance of transcriptional control because
they have excluded non transcribed and poorly transcribed genes. We demonstrate that after
correcting the non linear bias in protein abundance data, estimating the contribution of
molecule-specific measurement error, and making reasonable assumptions to take the
expression levels of all genes into account, mRNA levels explain ~65-82% of protein
expression levels. Using additional system wide data on the rates of mRNA and protein
degradation, we calculate that transcription explains ~51-75% of the variance in true protein
levels, RNA degradation explains ~7-14%, translation ~13-24%, and protein degradation
~5-11%. Our analysis provides a more accurate framework for considering the relative
quantitative importance of each step in gene expression. A preprint of our work is publicly
available at http://arxiv.org/abs/1212.0587.
Page 101
EMBO Conference Series: Protein Synthesis and Translational Control
66
Dissecting the roles of GW182-interacting proteins in miRNA-mediated gene
repression
Florian Aeschimann, Gert-Jan Hendriks
Friedrich Miescher Institute for Biomedical Research, Switzerland
Presenter: Florian Aeschimann
MicroRNAs (miRNAs) are small untranslated RNAs acting as post-transcriptional gene
regulators. In animals, miRNA target genes are repressed through translational inhibition and
mRNA degradation mediated by GW182 family proteins. Those are recruited by miRNA-bound
AGO proteins to target mRNAs, where they are thought to assemble a repression complex
including factors to inhibit translation and factors to enhance RNA degradation. We aim to
identify and characterize proteins of this repression complex in the nematode Caenorhabditis
elegans using genetic enhancer screens. Worms have two GW182 proteins called AIN-1 and
AIN-2. While ain-1 mutants display only slight developmental defects, ain-2 mutants seem to
be of wild-type phenotype. However, depletion of both AIN-1 and AIN-2 is lethal to the worm.
Our screens are designed to find factors that, when mutated or depleted, specifically enhance
developmental defects of ain-1 mutant worms. Hits from the screens are tested to be involved
in the miRNA pathway by measuring if they influence repression of miRNA target reporters in
living worms. Subsequently, we plan to test factors shown to affect miRNA activity for their
involvement in translational repression and/or mRNA degradation of endogenous miRNA
targets using the ribosome profiling technique.
Page 102
Poster Abstracts
67
Glycyl-tRNA synthetase is required for shifting Ribosome Landing Pad to
upstream AUG codon of PV IRES element
Dmitry Andreev, Ilya Terenin, Sergey Dmitriev, Ivan Shatsky
Moscow State University, Russian Federation
Presenter: Dmitry Andreev
The intriguing feature of picornavirus IRES elements of type I is the presence of conserved
upstream AUG codon. In case of poliovirus IRES, this putative initiation codon is located 157
nt upstream of the main AUG and is out of frame to the poliovirus genome reading frame.
However, it is known that mutation of this AUG result in inhibition, rather than stimulation of
translation of the main ORF. Here we investigated the effect of Glycyl tRNA synthetase (GARS),
the recently identified crucial ITAF required for picornavirus type I translation, on translation
initiation site selection. Surprisingly, GARS stimulates translation more efficiently when reporter
was fused directly with uAUG than for wt PV IRES. When uAUG was mutated the effect of
GARS becomes less prominent. Thus, GARS mediates loading of ribosomes to uAUG which in
turn results in increase of initiation on downstream main AUG. In order to understand how
GARS activates translation of PV IRES, cell free translation system was supplemented with
various translation initiation factors with or without GARS. eIF4G fragment p100 was able to
compensate the absence of GARS added. The shorter fragment of eIF4G (eIF4G MD) was also
able to stimulate translation from main AUG, but unexpectedly was strongly inhibitory to
translation driven from uAUG. We propose that GARS increases binding of eIF4G to PV IRES
and regulates eIF4G activity in order to allow the initiation on uAUG followed by transfer of
ribosome to the main AUG codon.
Page 103
EMBO Conference Series: Protein Synthesis and Translational Control
68
mRNA degradation on the ribosome in Drosophila cells
Sanja Antic1, Anna Skucha2, Silke Dorner1
1 Max F. Perutz Laboratories & University of Vienna, Austria
2 Center of Molecular Medicine, Austria
Presenter: Sanja Antic
The translation and degradation of mRNAs are two key steps in gene expression.
Consequently, both processes are highly regulated and targeted by many factors including
miRNAs. Even though translation and mRNA degradation are tightly coupled, it was suggested
that mRNA degradation occurs only after dissociation from the ribosomes in cytoplasmic
bodies, named P-bodies. Only recently the possibility of co-translational mRNA degradation in
yeast was discussed. However, at this point it is unclear whether mRNA degradation on the
ribosome would be limited to particular mRNA species or could also occur in multicellular
eukaryotes. We have investigated the possibility of co-translational mRNA degradation in
Drosophila cells. The co-purification of mRNA degradation factors with heavy fractions of
polysome profiles of cell lysates was an important first step. However, since P-bodies could
co-migrate with heavy polysome fractions we further established the affinity purification of
ribosomes from Drosophila S2 cell lysates. We could demonstrate the co-purification of various
deadenylation and decapping factors with ribosomes. Interestingly, also the factors of the
miRNA effector components, AGO1 and GW182, co-purify with ribosomes. Ongoing
experiments investigate the abundance of decapped mRNAs on ribosomes. In summary our
findings strongly suggest the ribosomes as an alternative site for mRNA degradation in
Drosophila.
Page 104
Poster Abstracts
69
Structural insights into drug-induced ribosomal stalling
Stefan Arenz, Otto Berninghausen, Roland Beckmann, Daniel N. Wilson
Gene Center Munich, LMU Munich, Germany
Presenter: Daniel N. Wilson
Expression of the resistance-gene ermB depends on ribosome stalling during translation of the
upstream ermB leader peptide (ermBL). Thereby, the sequence of the nascent polypeptide as
well as the presence of an inducing drug (e.g. erythromycin) are crucial for stalled-ribosome
-complex (SRC) formation (reviewed by 1). Here we report a cryo-electron-microscopy
structure at 4.5 Å resolution providing the structural basis for our model of how the stalling
signal is communicated from the ribosome exit tunnel, via a relay-mechanism, back to the
peptidyl-transferase center (PTC) in order to inactivate the A-site on the ribosome.
References:
[1] Vázquez-Laslop, N., Ramu, H., & Mankin, A. (2011). Nascent peptide-mediated ribosome
stalling promoted by antibiotics.
Page 105
EMBO Conference Series: Protein Synthesis and Translational Control
70
Novel proteomic approach reveals cell cycle specific fluctuations
in mRNA Translation
Orna Elroy-Stein, Ranen Aviner, Tamar Geiger
Tel Aviv University, Israel
Presenter: Orna Elroy-Stein
The ability to monitor, identity and quantity proteins produced under various conditions is
essential to our understanding of gene expression. Given that mRNA translation is the most
significant determinant of protein levels, knowledge of the cellular translatome and its response
to stress or stimuli has overwhelming implications on basic and clinical research. Ribosome
profiling is rapidly emerging as an important experimental tool. It does, however, suffer several
drawbacks, including intensive labor, high costs and reliance on mRNA measurement for
predicting protein levels. We have developed a simple and inexpensive proteomic approach for
direct identification and quantification of actively translated proteins, which we termed
PUromycin-associated Nascent CHain Proteome (PUNCH-P). This approach is readily
applicable to both cultured cells and whole tissues, and can therefore be used to study a
diverse spectrum of biological questions related to rapid changes in protein synthesis.
PUNCH-P is based on use of isolated polysomes for cell-free incorporation of biotinylated
puromycin into newly-synthesized proteins followed by affinity purification and mass
spectrometric (LC-MS/MS) analysis. We show that PUNCH-P analysis is rapid, reproducible
and equally as accurate as ribosome profiling. As a test case we used PUNCH-P to measure
cell cycle regulated changes in protein synthesis. We measured cell cycle-specific fluctuations
in synthesis for over 5,000 proteins in mammalian cells and identified hundreds of proteins that
are subject to differential regulation at the level of translation. Differentially synthesized proteins
include known regulators of cell cycle progression, as well as other proteins not previously
implicated in cell cycle regulation. Moreover, the data reveals insights related to global
translational regulation of specific sub-classes of mRNAs.
Page 106
Poster Abstracts
71
Elucidation of the determinants of IRES-mediated translation of cellular mRNAs
Eric Baggs, Bert Semler
Department of Microbiology and Molecular Genetics, University of California Irvine, United
States of America
Presenter: Eric Baggs
Internal ribosome entry sites (IRESs) are regions of mRNAs that facilitate direct binding of
components of the initiation complex for protein synthesis, independent of the cap and the
5’-terminus. While first discovered in viruses, there are several cellular mRNAs that harbor
these elements. The current study is evaluating the requirements (both canonical and
non-canonical) for translation initiation mediated by the voltage-gated potassium-channel
(Kv1.4) IRES as well as the lymphoid enhancer-binding factor 1 (LEF1) IRES. Kv1.4 is a shaker
related family member that contributes to the repolarizing phase of the cardiac action potential.
Expression of Kv1.4 is tissue-specific, in spite of its relatively non-specific promoter, and is
regulated at the post-transcriptional level. LEF1 is a transcription factor that mediates WNT
signaling, the alteration of which has been linked to colon cancer. Notably, there is a
discordance between LEF1 mRNA, LEF-1 protein, and WNT signaling in differentiating cells at
hair follicles, pointing to its regulation post-transcriptionally. Using both a genetic (yeast
3-hybrid screen) and a biochemical approach (biotinylated-RNA capture), we aim to discover
proteins that modulate the activity of these IRES elements. In addition to these targeted
approaches, we are using a deductive approach by altering cellular growth conditions and
evaluating the effects on translation from these two IRESs compared to that of cap-dependent
translation. Preliminary results suggest that factors or conditions related to cell cycle may play
a role in the efficiency of translation from both of these IRES elements when compared to 7
m-G cap driven translation. Our findings point to potential non-canonical translation factors
modulating the recruitment of ribosomes to these sites.
Page 107
EMBO Conference Series: Protein Synthesis and Translational Control
72
Elucidating mechanisms of translation with computational analysis of ribo-seq
data
Audrey Michel1, Patrick BF O'Connor1, K Roy Choudhury2, Andrew Firth3, Gene-Wei Li4,
Nicholas Ingolia5, Jonathan S Weissman4, John Atkins6, Pavel Baranov1
1 University College Cork, Ireland
2 Duke University, United States of America
3 University of Cambridge, United Kingdom
4 University of California, San Francisco, United States of America
5 Carnegie Institution for Science, United States of America
6 University of Utah/University College Cork, United States of America
Presenter: Pavel Baranov
Ribosome profiling ( ribo-seq ) is a recently developed technique that provide Genome Wide
Information on Protein Synthesis (GWIPS) in vivo . It is based on the deep sequencing of
ribosome protected mRNA fragments which allows the ribosome density along all mRNA
transcripts present in the cell to be quantified. Analysis of differential expression is an obvious
application of this technique that is rapidly gaining popularity. Here, however, we present the
results of our computational analysis of ribo-seq data that reveal various mechanistic aspects
of translation. By utilizing the triplet periodicity of ribosome footprints obtained in HeLa cells,
we have been able to detect human mRNAs with regions translated in reading frames
alternative to the predicted. Our analysis of ribo-seq data in bacteria suggests that ribosome
interactions with Shine-Dalgarno sites lead to the accumulation of extra mRNA in the ribosome
mRNA channel. We also present a computational model for calculating the efficiencies of
individual translation initiation sites (TISs) from the footprinting of initiating ribosomes. The
model is based on the leaky scanning mechanism of translation initiation. Application of our
model demonstrated that despite the high frequency of non-AUG TISs revealed with ribo-seq ,
the probability of initiation at non-AUG codons is considerably lower than at AUG codons. This
suggests that initiation at most non-AUG codons contributes to the noise of gene expression
rather than to the generation of protein products with functions that are distinct from AUG
initiated products. Our model of leaky scanning provides a simple and efficient method for
evaluating the strength of individual initiation codons based on ribo-seq data.
Page 108
Poster Abstracts
73
Post-transcriptional regulation of gene expression is essential for the cellular
response to cold stress
Amandine Bastide1, John R.P. Knight1, Diego Peretti1, Ruth Spriggs2, Thomas Jakson1,
Xavier Pichon1, Joanne Roobol3, Martin Bushell1, Mark Smales4, Giovanna Mallucci1, Anne
Willis1
1 MRC, Toxicology Unit, United Kingdom
2 The University of Leicester, United Kingdom
3 School of Biosciences, University of Kent, United Kingdom
4 Centre for Molecular Processing and Protein Science Group, United Kingdom
Presenter: Amandine Bastide
Cooling is used extensively in patho-physiological situations; however the genome-wide
changes that occur following a reduction in ambient temperatures are not well described. We
have carried out transcriptional, translational and miRNA profiling of cooled cells and show that
control of protein synthesis and reprogramming of translation make the major contribution in
the response to this stress. Our data show that alterations in translation are mediated by an
increase in the phosphorylation of eIF2 alpha, a decrease in elongation rates and, very
surprisingly, in a reduction in the abundance of the 40S ribosomal subunits. Overall these
changes result in an attenuation of protein synthesis and a selective increase in synthesis of
proteins that allow the cells to respond to the cold stress including chaperones and ER
resident proteins. We show that selective synthesis of these proteins is mediated by elements
in the 5’UTR of their corresponding mRNAs. Our data suggest that modulation of the proteins
that control the cold-stress response could provide new ways in which to induce the protective
effects of temperature reduction without cooling.
Page 109
EMBO Conference Series: Protein Synthesis and Translational Control
74
Role of eIF5 phosphorylation in translation regulation
Ira Bavli-Kertselli, Daniel Melamed, Lavi Bar-Ziv, Yoav Arava
Technion, Israel
Presenter: Ira Bavli-Kertselli
Cells respond to various intra- and extra-cellular stimuli by changing their protein repertoire.
This ability is mediated in most cases by signal transduction pathways and phosphorylation
events that regulate the gene expression program. At the translation level, several regulatory
pathways have been characterized, yet for many stimuli the regulatory factors are unknown.
Glucose is one of the major carbon sources for S.cerevisiae and mediates many signaling
cascades. Abrupt glucose removal was shown to induce the fastest and strongest translation
inhibition in yeast cells. Neither TOR nor eIF2a are required for this inhibition. Intriguingly, there
are many mutant yeast strains in which this arrest does not occur. Yet none of these carry a
mutation in a core translation factor. We sought that the resistant phenotype of these strains is
due to altered expression level of a translation factor, which thereby change the cells' ability to
sense stress. Indeed, only few hours of growth under low glucose conditions render cells
resistant to glucose depletion, consistent with an outcome of gene expression changes rather
than genomic mutation. Using genome-wide expression analysis we identified several
translation factors with modified expression profiles in mutant cells. Directed overexpression of
one of these factors (eIF5) appeared to rescue the stress-resistant phenotype of a mutant
strain. Deletion analysis indicated that full rescue necessitated multiple domains from eIF5, yet
the regulatory phosphorylation site in position 397 was not necessary. Mass spectroscopy
analysis of wild-type cells revealed that only Threonine 191 becomes highly phosphorylated
upon glucose depletion. This site appeared to be important for regulation because, contrary to
overexpression of normal eIF5, T191A mutant was not able to rescue a resistant phenotype.
Taken together, these results suggest that phosphorylation of eIF5 in a novel regulatory site
mediates translation upon glucose depletion.
Page 110
Poster Abstracts
75
eIF6 activation and inhibition control tumorigenesis
Stefano Biffo1, Marilena Mancino1, Simone Gallo2, Elisa Pesce1, Piera Calamita3, Stefania
Oliveto1
1 University of Eastern Piedmont, Italy
2 Università Vita-Salute, Italy
3 San Raffaele, Italy
Presenter: Stefano Biffo
eIF6 prevents improper 80S ribosomal association in the absence of mRNA. In the past, we
have shown that the antiassociation activity of eIF6 is necessary for tumor progression, both in
vitro and in vivo. Here we show that several tumors overexpress hyperphoshorylated eIF6. eIF6
phosphorylation in the C-terminus is controlled by several stimuli, and its impairment results in
reduced tumor growth. A HTS assay for screening eIF6 antagonists has been developed, in
order to identify eIF6 blockers. Taken together data suggest that eIF6 is necessary for tumor
progression and its targeting is feasible and results in tumor regression.
Page 111
EMBO Conference Series: Protein Synthesis and Translational Control
76
The natural suppressor tRNAs are differently incorporated during stop codon
readthrough in eukaryotes
Sandra Blanchet1, Manuela Argentini2, David Cornu2, Olivier Namy3
1 Institut de Genetique et Microbiologie, France
2 SiCAPS, France
3 CNRS, France
Presenter: Sandra Blanchet
Translation termination occurs when a stop codon enters the ribosomal A site where it is
recognized by the eukaryotic translation termination factor eRF1. In cells, termination always
competes with readthrough, which corresponds to the incorporation of a near-cognate tRNA
at the stop codon. This allows translation to continue in the same frame. We aim to identify
natural suppressor tRNAs inserted at a stop codon during readthrough. That’s why I
developed a system in Saccharomyces cerevisiae to express and purify the Glutathione
S-transferase, carrying a stop codon, 4 codons downstream the start codon of the open
reading frame. Readthrough proteins are then analyzed by mass spectrometry to identify
amino acids incorporated at the readthrough site. We have already tested the three stop
codons located in the same nucleotide sequence and identified the amino acids. We have also
tested the influence of antibiotics, used in therapeutic approaches for premature stop codon
diseases to increase the level of stop codon suppression. We found that the molecules
inducing readthrough have an impact on the proportion of the amino acids found at the stop
codon. This has important therapeutics implications because the activity of the protein
re-expressed by readthrough can be strongly modified depending on the amino acid
incorporated during suppression. We now aim to test the influence of the stop codon
nucleotide context on the nature or proportion of the amino acids inserted, in order to make
predictive rules of incorporation. These data could help to personalize the treatment for
premature stop codon diseases.
Page 112
Poster Abstracts
77
Substrate specificity of scavenger decapping enzymes (DcpS) towards
dinucleotide cap analogs modified within 7-methylguanine moiety
Elzbieta Bojarska, Karolina Piecyk, Marzena Jankowska-Anyszka, Maciej Lukaszewicz,
Zbigniew Darzynkiewicz, Janusz Stepinski, Edward Darzynkiewicz
University of Warsaw, Poland
Presenter: Elzbieta Bojarska
Decapping scavengers are involved in eukaryotic mRNA degradation process. They catalyze
the cleavage of residual cap structure m 7 GpppN or short capped oligonucleotides resulting
from exosom-mediated 3’ to 5’ digestion of deadenylated transcript. Hydrolysis of cap
structure releases m 7 GMP and ppN or diphosphate terminated oligo mRNA containing less
than 10 nucleotides. DcpS enzymes are members of the HIT family of pyrophosphatases with
a conserved histidine triad in the active site. For the specific cap recognition by DcpS enzymes
the positive charge is required, introduced by a substituent at N7 position of guanine moiety,
as well as the triphosphate chain. The type of modification of the second nucleoside does not
influence on the kinetic parameters of hydrolysis. Moreover, the second nucleoside is not
necessary for DcpS activity. To further characterize molecular determinants for the efficient
cap-binding and hydrolysis, we examined the influence of alkyl and benzyl substituents
introduced at N2 or N7 position of guanine moiety of cap analogs on their substrate properties
towards DcpS proteins. Enzymatic cleavage of modified compounds (et 7 GpppG, b 7
GpppG, ib 7 GpppG, bn 7 GpppG, ( p Clbn) 7 GpppG, m 7 et 2 GpppG, m 7 b 2 GpppG)
catalyzed by human and nematode decapping scavengers was tested by means of
fluorescence and HPLC-based assays. Kinetic analysis of DcpS-mediated hydrolysis indicates
that investigated enzymes accept as substrates the dinucleotides bearing ethyl, butyl, benzyl
and p -Cl-benzyl at N7, but not at N2 position. Cap analog, substituted by isobutyl at N7
position, occurred to be a poor substrate for nematode DcpS, and resistant to human enzyme.
Our data indicate the crucial role of N2 position for cap recognition and efficient hydrolysis,
contrary to the N7 position, which could be modified by differently sized substituents.
Work supported by a grant 02/EuroNanoMed/2011.
Page 113
EMBO Conference Series: Protein Synthesis and Translational Control
78
Doing things differently: The universally conserved translational GTPase HflX
reveals a new mode of GTPase activation on the ribosome
Harland Brandon, Mackenzie L. Coatham, Jeffrey J. Fischer, Hans-Joachim Wieden
University of Lethbridge, Canada
Presenter: Harland Brandon
Ribosome dependent protein synthesis is a highly regulated and accurate process essential for
every living cell. Besides the ribosome, an increasing number of protein factors are identified to
be required for robust protein synthesis in vivo. Several of these factors are so called molecular
switches that bind and hydrolyze guanosine triphosphate (GTP) in order to perform their
function, typically recognizing a certain functional state of the ribosome. In particular,
elongation factor (EF) Tu and G perform vital functions during translation and are stimulated by
the GTPase activating center (GAC) and Sarcin-Ricin loop (SRL) located on the large ribosomal
subunit. HflX is a universally conserved GTPase whose precise cellular function still remains
elusive. We have recently shown that HflX likely belongs to the group of translational GTPases,
targeting the ribosome which in turn is able to specifically regulate the GTPase ativity of HflX (1,
2). Here we report, that unlike other GTPases, HflX does not bind to the GAC / SRL region of
the ribosome. Using cross-linking, mass spectrometry, and primer extension analysis, we have
determined for the first time the HflX binding site on the ribosome. Our finding suggests that
the ribosome possesses a second GTPase activating centre distinct from the
well-characterized GAC / SRL.
References:
(1) Fischer, J.J., Coatham, M.L., Eagle Bear, S., Brandon, H.E., De Laurentiis, E.I., Shields,
M.J., Wieden, H.-J. (2012) Biochimie 94(8): 1647-1659. (2) Shields, M., Fischer, J., Wieden,
H.-J. (2009) Biochemistry 48: 10793-10802.
Page 114
Poster Abstracts
79
Characterisation of mammalian PABP4 expression and function
Matthew Brook, Hannah Burgess, Ross Anderson, William Richardson, Joao Sousa
Martins, Nicola Gray
University of Edinburgh, United Kingdom
Presenter: Matthew Brook
Poly(A)-binding protein 4 (PABP4, PABPc4) is a vertebrate PABP-family member. PABP1, the
prototypical member, enhances global translation and can promote or repress mRNA-specific
translation. PABP1 also has a variety of roles in regulating global and mRNA-specific turnover.
However the function of mammalian PABP4 remains largely unknown, thus we are examining
its molecular and physiological roles. Importantly, by establishing the tissue- and cell-specificity
of mouse PABP4 and PABP1 expression, we find that they have distinct, but overlapping,
patterns of expression. PABP4 and PABP1 both appear to be widely expressed, in contrast to
ePABP and tPABP whose expression is highly restricted, with many mammalian cell-types
apparently expressing multiple PABP proteins. Furthermore, specific cell types within a tissue
can express divergent PABP subsets, indicating highly coordinated expression of PABP family
members. We find that PABP4, in contrast to PABP1, has the potential to be expressed as
multiple isoforms in mouse (and human), with the exons encoding the proline-rich linker region
(PRLR) between the RNA-recognition motifs and the PABC domain being subject to complex
alternative splicing. Consistent with this, we observe differential expression of PABP4 isoforms
between a panel of mouse tissues. The PABP1 PRLR is required for homo-dimerisation but
the function(s) of the highly divergent PABP4 PRLR(s) are unknown. However, all PABP4
isoforms tested thus far efficiently bind poly(A) RNA. Studies to examine the regulation of
translation by PABP4, to define the PABP4-specific interactome and to map PABP4
post-translational modifications will also be discussed.
Page 115
EMBO Conference Series: Protein Synthesis and Translational Control
80
Impact of methylations of m2G966/m5C967 in 16S rRNA on bacterial fitness
and translation initiation
Dmitry Burakovskiy1, Irina Prokhorova2, Petr Sergiev3, Pohl Milón1, Olga Sergeeva3, Alexey
Bogdanov3, Marina Rodnina1, Olga Dontsova3
1 MPI for Biophysical Chemistry, Germany
2 IGBMC, France
3 Lomonosov Moscow State University and A.N. Belozersky Institute of Physico-Chemical
Biology, Russian Federation
Presenter: Dmitry Burakovskiy
The functional centers of the ribosome in all organisms contain rRNA modifications, which are
introduced by specialized enzymes and come at an energy cost for the cell. Surprisingly, none
of the modifications tested so far was essential for growth and hence the functional role of
modifications is largely unknown. Here we show that the methyl groups of nucleosides m 2
G966 and m 5 C967 of 16S rRNA in Escherichia coli are important for bacterial fitness. In vitro
analysis of all phases of translation suggests that the m 2 G966/m 5 C967 modifications are
dispensable for elongation, termination, and ribosome recycling. Rather, the modifications
modulate the early stages of initiation by stabilizing the binding of fMet-tRNA fMet to the 30S
pre-initiation complex prior to start-codon recognition. We propose that the m 2 G966 and m 5
C967 modifications help shaping the bacterial proteome, most likely by fine-tuning the rates
that determine the fate of a given mRNA at early checkpoints of mRNA selection.
Page 116
Poster Abstracts
81
Regulation of mammalian GCN2 by GCN1 and IMPACT
Tavane Cambiaghi1, Catia Pereira1, Michael Bolech2, Renuka Shanmugam2, Evelyn
Sattlegger2, Beatriz Castilho1
1 Unifesp, Brazil
2 INMS, Massey University, New Zealand
Presenter: Tavane Cambiaghi
General protein synthesis is largely regulated by the availability of the ternary complex
eIF2-GTP-Met.tRNAi which mediates one of the earliest steps of translation. The
phosphorylation of the alpha subunit of eIF2 impedes the formation of this complex, inhibiting
general protein synthesis but increasing the translation of specific messages, such as GCN4 in
yeast and ATF4 in mammals. GCN2, one of the four mammalian eIF2α kinases, is activated by
amino acid starvation, proteasome inhibition, glucose deprivation and UV irradiation. We have
previously shown that IMPACT, a protein highly expressed in neurons, binds to GCN1, a
GCN2 effector protein, and inhibits GCN2 activation under amino acid starvation conditions.
Here, we provide evidence that IMPACT affects specifically the activation of GCN2 under all
stress conditions known to activate this kinase in mammalian cells. Accordingly, we show that
mammalian GCN2 requires GCN1 for activation and that IMPACT competes with GCN1 for
GCN2 binding. We also demonstrate that IMPACT is capable of inhibiting the activation of
Gcn2 in yeast cells subjected to a variety of stress conditions. Our data provide evidence that
mammalian GCN2 functions as its yeast counterpart, and that mammalian and yeast IMPACT
provide identical mechanisms for controlling the activation of GCN2 in eukaryotes.
Page 117
EMBO Conference Series: Protein Synthesis and Translational Control
82
HuR and miR-1192 respectively promote and reduce myogenesis by
modulating the translation of HMGB1 mRNA
Anne Cammas1, Virginie Dormoy-Raclet2, Barbara Celona3, Xian Jin Lian2, Kate van der
Giessen2, Marija Zivojnovic2, Silvia Brunelli4, Francesca Riuzzi5, Guglielmo Sorci5, Brian
Wilhelm6, Sergio DiMarco2, Rosario Donato5, Marco E. Bianchi3, Imed-Eddine Gallouzi2
1 INSERM, France
2 McGill, Canada
3 San Raffaele University and Research Institute, Italy
4 University of Milan-Bicocca, Italy
5 University of Perugia, Italy
6 Université de Montréal, Canada
Presenter: Anne Cammas
Upon muscle injury the high mobility group box 1 (HMGB1) protein is up- regulated and is
secreted to initiate reparative responses. Here we show that the expression level of HMGB1
controls myogenesis, and a 50% reduction severely hinders myogenesis in vitro, during
embryo development and after adult muscle injury. HMGB1 expression in muscle cells is
regulated at the translational level: the miRNA miR-1192 inhibits HMGB1 translation and the
RNA-binding protein HuR promotes it. HuR binds to a cis-element, HuRBS, located in the
3'UTR of HMGB1 transcript, and miR-1192 is recruited at the same time to a seed element
adjacent to the HuRBS. When HuR is bound to the HuRBS, miR-1192 is unable to inhibit
HMGB1 translation due to its failure to recruit Argonaute 2 (Ago2). Depleting HuR reduces
myoblast fusion, and silencing miR-1192 re-establishes the fusion potential of HuR-depleted
cells. Therefore, our data support a model whereby HuR promotes the commitment of
myoblasts to myogenesis by enhancing the translation of HMGB1 and suppressing the
translation inhibition mediated by miR-1192.
Page 118
Poster Abstracts
83
General and differential changes in the translatome participate in the
establishment of the heat stress response in Arabidopsis seedlings
Mar M Castellano1, Emilio Yángüez1, Ana B. Castro-Sanz1, Nuria Fernández-Bautista2
1 INIA, Spain
2 UPM, Spain
Presenter: Mar M Castellano
Heat stress is one of the most prominent and deleterious environmental threads affecting plant
growth and development. Upon high temperatures, plants launch specialized gene expression
programs that promote stress protection and survival. These programs involve global and
specific changes at the transcriptional and translational levels; however the coordination of
these processes and their specific role in the establishment of the heat stress response is not
fully elucidated. We have carried out a genome wide analysis to monitor simultaneously the
individual changes in the transcriptional and translational mRNA levels of Arabidopsis thaliana
seedlings after the exposure to a heat shock stress. Our results demonstrated that,
superimposed to transcription, translation exerts a wide but dual regulation of gene
expression. For the majority of the mRNAs, translation is severely repressed causing a deep
decrease in the association of the bulk of mRNAs to polysomes. However, some relevant
mRNAs involved in different aspects of homeostasis maintenance follow a differential pattern of
translation. Analysis of the sequence of the differentially translated mRNAs unraveled some
special features that take part in the discrimination mechanisms for mRNA polysome loading.
Among the identified differential translated genes stand out key regulators of the stress
response highlighting the main role of translation in the early establishment of physiological
response of plants to elevated temperatures.
Page 119
EMBO Conference Series: Protein Synthesis and Translational Control
84
The translational repressors Caf20p and Eap1p are associated with specific
transcripts and actively translating ribosomes
Lydia Castelli, Joe Costello, Chris Kershaw, William Rowe, Paul Sims, Simon Hubbard,
Chris Grant, Mark Ashe, Graham Pavitt
University of Manchester, United Kingdom
Presenter: Lydia Castelli
One protein synthesis control pathway involves eIF4E binding proteins (4E-BPs), which inhibit
translation by binding and sequestering the 5’ cap binding protein eIF4E away from its partner
eIF4G. The inhibition of translation initiation through 4E-BPs can occur in either a non-specific
(e.g. 4E-BP1) or an mRNA specific manner through interaction with RNA elements or partner
RNA binding proteins (e.g. Maskin or Cup). Prior microarray analysis showed that individual
deletion of the two yeast 4E-BPs, caf20Δ and eap1Δ, alters the polysome association of
different but overlapping subsets of mRNAs. Therefore, there is some mRNA specificity
underlying their regulation of translation. To identify mRNAs directly bound by each 4E-BP we
have taken a RIP-Seq approach using TAP-tagged factors. This has identified a large cohort of
target mRNAs for each 4E-BP, that includes many transcription factors, suggesting links
between different stages of gene regulation. In order to assess the mechanisms by which the
yeast 4E-BPs are targeted to different mRNAs, the protein interactions of Caf20p and Eap1p
were investigated using TAP and FLAG purified factors and mass spectrometry. Both proteins
unexpectedly associated with many ribosomal proteins. The 4E-BPs are distributed across
polysome gradients in a manner similar to many translation initiation factors rather than that
expected for complexes containing inhibited mRNAs. Ribosome association was not
dependent on the eIF4E interaction motif and is disrupted by high salt conditions showing that
they are not core ribosomal subunits. Taken together, these results suggest that the yeast
4E-BPs bind a large number of target mRNAs, but don’t behave as simple translation
repressors. They either have a separate ribosome associated function or may bind to
translating ribosomes, poised to regulate translation under specific conditions or on specific
mRNAs.
Page 120
Poster Abstracts
85
New in vivo RNA-binding architectures discovered by RBDmap
Alfredo Castello1, Bernd Fischer1, Sophia Foehr1, Anne-Marie Alleaume1, Tomaz Curk2,
Jeroen Krijgsveld1, Matthias Hentze1
1 EMBL Heidelberg, Germany
2 University of Ljubljana, Slovenia
Presenter: Alfredo Castello
The RNA interactomes of HeLa and HEK293 cells jointly comprise 1106 RNA-binding proteins
(RBPs) (1, 2), with almost half of these lacking well-defined RNA-binding domains (RBDs),
suggesting the existence of numerous unknown RNA-binding architectures. Here, we report
RBDmap, a new method built on interactome capture (3), to comprehensively identify the
RBDs of native RBPs in proliferative HeLa cells. Making use of in vivo UV-crosslinking of RBPs
to polyadenylated RNAs, capture on oligo(dT) magnetic beads, proteolytic mapping and mass
spectrometry combined with a sophisticated scoring algorithm, RBDmap “re-discovered” the
known RNA-binding sites (e.g. RRM, KH) of numerous well characterized RBPs, validating the
approach. Strikingly, RBDmap identified dozens of additional RNA-binding architectures (e.g.
thioredoxin, SSB, RAP, WD40) in multiple non-homologous proteins, also including disordered
motifs such as basic patches. RBDmap thus instructs on the modes of RNA-binding of
hundreds of proteins in their native cellular states, providing valuable structural and functional
insights into RNA biology. For example, the identification of the thioredoxin domain as a
high-confidence RBD reveals an intriguing link between the redox state of cells and RNA
metabolism.
References:
[1] A. G. Baltz et al., Mol Cell 46, 674 (2012). [2] A. Castello et al., Cell 149, 1393 (2012)
[3] A. Castello et al., Nat Protoc 8, 491 (2013).
Page 121
EMBO Conference Series: Protein Synthesis and Translational Control
86
Impact of rRNA methyl-transferase Fibrillarin down-regulation on ribosome
synthesis and ribosome intrinsic activity
Stéphane Belin1, Sandra Ghayad1, Sabine HACOT1, Peter Warren2, Judith Steen2,
Jean-Jacques DIAZ1, Frederic Catez1
1 Cancer Research Center of Lyon, France
2 Children's Hospital Boston, United States of America
Presenter: Frederic Catez
A growing body of evidence support that ribosome composition (ribosomal proteins and RNAs)
plays a direct role in the intrinsic translational activity of the ribosome and in translational
control. Biochemical and structural data have demonstrated that ribosomal RNA (rRNA) are
ribozymes and are directing the translation process. rRNAs carry chemical modifications: base
and ribose methylation and pseudourydilation. 2'-O-ribose methylation is the most abundant
modification with 105 sites identified in human rRNAs. Ribose-methylation has been shown to
affect translational fidelity and translation initiation through internal ribosome entry sites (IRES).
However, the precise role of rRNA methylation in ribosome function and in translational control
remains unclear, and the consequences of rRNA methylation alteration on cellular phenotype
are poorly known. We recently showed that fibrillarin (FBL), the ribose methyl-transferase of
rRNAs, is differentially expressed in cancer cells, and that increased FBL expression is
associated with increased rRNA methylation, higher IRES dependent translation initiation, and
lower translational fidelity (Belin, S. et al. PLoS ONE, 2009 ; Gahyad, S. et al. submitted). In this
study, we down regulated FBL expression in HeLa cells to further evaluate the impact of FBL
expression on ribosome composition and activity. Here we show that FBL down regulation
alters ribosome production. Analysis of purified ribosomes by northern blot, RT-qPCR and
proteomic approaches revealed modifications in ribosome composition. rRNA methylation is
globally decreased, with a differential site-by-site alteration. Ribosome translational capacity
was explored using reporter assays and revealed a higher translational fidelity and a lower IRES
dependent translation. Overall our data shows that change in FBL expression alters ribosome
quality and activity, and supports that FBL expression in normal and pathological tissues might
participate in translational control.
Page 122
Poster Abstracts
87
Modifiying Chemotherapy Response by Targeted Inhibition of Eukaryotic
Initiation Factor 4A
Regina Cencic1, Francis Robert1, Gabriela Galicia Vazquez1, Abba Malina1, Kontham
Ravindar2, Ragan Somaiah2, Philippe Pierre3, Junichi Tanaka4, Pierre Deslongchamps2,
Jerry Pelletier1
1 McGill University, Canada
2 Université Laval, Canada
3 Université de la Méditerranée, France
4 University of the Ryukyus, Japan
Presenter: Regina Cencic
The recruitment of the 40S ribosomal subunit and associated factors to the mRNA template
during translation initiation is a highly regulated process. It is under regulation of eukaryotic
initiation factor (eIF) 4F – a heterotrimeric complex comprising of eIF4E (a cap-binding protein),
eIF4A (a DEAD box RNA helicase), and eIF4G (a large scaffolding protein). Translation initiation
is frequently deregulated in human tumors due to increased activity of regulatory upstream
signalling kinase pathways (PI3K and MAPK) that influence eIF4F assembly and activity.
Hence, there is much interest in targeted therapies that block eIF4F activity to assess the
consequences on tumor cell growth and chemotherapy response. Our lab has previously
identified the natural product hippuristanol, an inhibitor of translation initiation that prevents
eIF4A from interacting with RNA through an allosteric mechanism of action. Hippuristanol is a
selective inhibitor of eIF4AI and eIF4AII that functions in vitro and in vivo to block translation
initiation. Utilizing a Myc-driven tumor model (the Em-myc mouse – a transgenic model of
Non-Hodgkin’s lymphoma), we have previously shown that altering translation initiation can
dramatically impact on tumor initiation and maintenance. We present data demonstrating that
hippuristanol is capable of sensitizing Em-myc tumor cells to standard of care agents. As well,
we find that treatment of cells with hippuristanol leads to a rapid decline in Mcl-1 levels, and
ectopic over-expression of Mcl-1 alters sensitivity to hippuristanol. Since Mcl-1 levels are
significantly affected by hippuristanol, combining its use with the Bcl-2 family inhibitor,
ABT-737 leads to a potent synergistic response in triggering cell death in mouse and human
lymphoma and leukemia cells. Taken together our results highlight eIF4AI as a therapeutic
target for modulating tumor cell response to chemotherapy.
Page 123
EMBO Conference Series: Protein Synthesis and Translational Control
88
Ribosome profiling reveals resemblance between long non-coding RNAs and
5’ leaders of coding RNAs
Guo-Liang Chew1, Andrea Pauli1, John Rinn1, Aviv Regev2, Alexander Schier1, Eivind Valen1
1 Harvard University, United States of America
2 Massachusetts Institute of Technology, Howard Hughes Medical Institute, United States of
America
Presenter: Guo-Liang Chew
Large-scale genomics and computational approaches have identified thousands of putative
long non-coding RNAs (lncRNAs). It has been controversial, however, as to what fraction of
these RNAs is truly non-coding. Here we combine ribosome profiling with a machine-learning
approach to validate lncRNAs during zebrafish development in a high throughput manner. We
find that dozens of proposed lncRNAs are protein-coding contaminants and that many
lncRNAs have ribosome profiles that resemble the 5’ leaders of coding RNAs. Analysis of
ribosome profiling data from ES cells reveals similar properties for mammalian lncRNAs. These
results clarify the annotation of developmental lncRNAs and suggest a potential role for
translation in lncRNA regulation. In addition, our computational pipeline and ribosome profiling
data provides a powerful resource for the identification of translated open reading frames
during zebrafish development.
Page 124
Poster Abstracts
89
OGFOD1 is a novel ribosomal prolyl hydroxylase involved in translational
control and stress granule formation
Rachelle Singleton1, Phebee Liu-Yi1, Fabio Formenti1, Wei Ge1, Roman Fischer1, Atsushi
Yamamoto1, Mathew Coleman1, Mukaram Mackeen1, Julie Adam1, Patrick Pollard1, Benedikt
Kessler1, Pablo Wappner2, Christopher J. Schofield1, Peter Ratcliffe1, Matthew Cockman1
1 University of Oxford, United Kingdom
2 Fundacion Instituto Leloir, Argentina
Presenter: Matthew Cockman
OGFOD1 (2-oxoglutarate and Fe (II) dioxygenase 1) is a conserved member of the
2-oxoglutarate dependent dioxygenase family of enzymes. Orthologs in yeast have been
implicated in diverse cellular functions ranging from oxygen-dependent regulation of sterol
response genes (Ofd1, S.pombe) to translation termination and mRNA polyadenylation (Tpa1,
S.cerevisiae). However, neither the biochemical activity of OGFOD1 nor the identity of its
substrate has been defined. Here we show that OGFOD1 is a prolyl hydroxylase that catalyses
the post-translational hydroxylation of a highly conserved residue (Pro-62) in the small
ribosomal protein rpS23. Unusually OGFOD1 retained a high affinity for, and formed a stable
complex with, the hydroxylated rpS23 substrate. Knockdown or knockout of OGFOD1
resulted in cell-type dependent induction of stress granule formation, translational arrest and
impairment of growth, which was complemented by wild-type but not catalytically inactive
OGFOD1. The work defines a new type of posttranslational prolyl hydroxylation with a role in
stress granule formation and translational control in mammalian cells.
Page 125
EMBO Conference Series: Protein Synthesis and Translational Control
90
Modelling acquired resistance to mTOR kinase inhibitors
Claire Cope1, Kathryn Balmanno1, Rebecca Gilley1, Paul Smith2, Sylvie Guichard2, Simon
Cook1
1 The Babraham Institute, United Kingdom
2 AstraZeneca, United Kingdom
Presenter: Simon Cook
The serine/threonine protein kinase mTOR represses autophagy and promotes mRNA
translation, protein synthesis and cell growth. mTOR acts downstream of signalling pathways
that are mutated in cancer making it an attractive target for new therapeutics such as the ATP
competitive mTOR kinase inhibitor AZD8055. To anticipate how tumour cells adapt to inhibition
of mTORC1 and mTORC2 we have generated AZD8055-resistant versions of SW620
colorectal cancer cells in vitro by chronic drug exposure. Acquired resistance was not due to
up-regulation of their driving KRAS oncogene or changes in ERK1/2 or AMPK signalling.
Rather, SW620:8055R cells exhibited a significant increase in eIF4E expression and
cap-dependent protein translation compared to parental cells and siRNA-mediated eIF4E
ablation resensitised these cells to the growth inhibitory effects of AZD8055. Our results
suggest that one mechanism by which tumour cells may develop resistance to mTOR inhibition
is by up-regulation of eIF4E and cap-dependent translation, highlighting the importance of this
pathway downstream of mTOR.
Page 126
Poster Abstracts
91
Targeted identification and purification of TOP mRNA translational regulators
Amy Cooke, Matthias Hentze
EMBL Heidelberg, Germany
Presenter: Amy Cooke
The synthesis of many proteins of the translational apparatus is selectively regulated in a
growth-dependent manner at the translational level. This was first observed over 3 decades
ago for messenger (m)RNAs that encode ribosomal proteins. These mRNAs contain a 5’
terminal oligopyrimidine tract (5’ TOP) that is required for proper regulation, thus they are
termed TOP mRNAs. The phenomenon of TOP-mediated translational control has been
well-studied and important advances on signalling pathways and functionally relevant features
of the TOP element have been made. However, it is still debated what trans-acting factors
regulate TOP mRNAs. This has been in large part due to technical challenges of isolating
specific RNAs with their bound protein partners. Recent advances in UV crosslinking, RNA
capture and proteomic methods allow “old problems” to be approached from new angles. We
are combining these recent advances to develop an in vitro system to identify
proliferation-dependent TOP regulators with the ultimate goal of characterizing their biological
function and mechanism. To this end, luciferase reporter constructs that recapitulate
TOP-mediated translational control have been generated. A 4-thio UTP labelled TOP luciferase
RNA generated by splint ligation will be added to these extracts. We will then apply
Photoactivatable-Ribonucleoside-Enhanced Crosslinking (PAR-CL) followed by RNA capture
and quantitative proteomics to identify TOP RNA-binding proteins (RBPs) (Castello et al., 2012;
Castello et al., 2013; Hafner et al., 2010). Taken together, this approach aims to identify TOP
RBPs and to develop an in vitro system to functionally characterize the candidates, which is a
long-standing question in the field of translational control.
References:
Castello A. et al., Cell 2012, 149 (6) Castello A. et al., Nat Protoc. 2013, 8 (3) Hafner M. et al,
Cell 2010, 141 (1)
Page 127
EMBO Conference Series: Protein Synthesis and Translational Control
92
The role and prevalence of non-canonical translation initiation codons in
generating mitochondrially-localised proteins
Joanne Cowan, Mike Allwright, Hayley Herbert, Meowea Hezwani, Richard Edwards, Mark
Coldwell
University of Southampton, United Kingdom
Presenter: Mark Coldwell
99% of proteins with a role in mitochondrial processes are synthesised from messages
transcribed from the nuclear genome. These mRNAs are translated in the cytoplasm and
require post-translational mechanisms to target them to the mitochondria. The sequence most
commonly required for targeting to this organelle is found in the N-termini of proteins, forming
an amphipathic α-helix which presents hydrophobic amino acids on one surface and positively
charged amino acids on the other. These respectively facilitate binding to TOM20 and TOM22,
subunits of the translocase of the outer mitochondrial membrane. In recent years, we have
been endeavouring to uncover situations where translation begins from initiation codons other
than the canonical AUG. We have used bioinformatic techniques to identify N-terminal
extensions in several proteins, which have then been confirmed by laboratory work. In one
such case, we have found isoforms of RPP25 that are translated from upstream GUG and
CUG codons that result in the relocalisation of the protein to mitochondria and the nucleus
compared to the AUG-initiated isoform. We will discuss our latest findings on the regulation of
initiation codon selection in this mRNA and the possible role of the novel forms of RPP25.
Given that the predicted mitochondrial targeting peptide (MTP) of RPP25 was discovered in a
region of the transcriptome annotated as 5’ UTR, we have undertaken further bioinformatic
work in an attempt to predict the presence of non-canonical initiation and MTPs in the 5’ UTRs
of other mRNAs termed as ‘mitochondrial’ according to their Gene Ontology (GO) term. Our
work suggests that several other mitochondrial proteins may use non-AUGs for initiation and
contain MTPs in regions currently annotated as untranslated. This work is funded by the
BBSRC.
Page 128
Poster Abstracts
93
RNAseq analysis of total and polysomal transcript populations in the MCF7
and MCF10A cell lines demonstrates extensive cell-specific mRNA 5’leader
heterogeneity
Joseph Curran1, Pascale Jaquier-Gubler2, Sergey Nikolaev3, Stylianos Antonarakis3, Patrick
BF O'Connor4, Pavel Baranov4
1 University of Geneva Medical School, Switzerland
2 Dept. of Microbiology and Molecular Medicine, University of Geneva Medical School,
Switzerland
3 Dept. of Genetic Medicine and Development, University of Geneva Medical School,
Switzerland
4 University College Cork, Ireland
Presenter: Joseph Curran
The mRNA 5’leader is a key element in translational regulation. This occurs at two levels,
referred to as quantitative and qualitative control. In the former, changes in the mRNA 5’leader
alters the amount of protein translated, whereas in the latter, changes in start site selection
occur. Changes arise due to the use of alternative promoters and/or alternative splicing.
Estimates of the number of genes with alternative 5’ ends vary from 12-22%, while estimates
of alternative promoter usage range from 10-18%. Conservation across species of elements
that play a role in translational control, suggest that these variations are functionally relevant. It
is therefore evident that the aberrant use of alternative promoters, that in-turn, alters the
translational read-out, may impact significantly on normal cellular function. MCF-10A are
recognized as ‘‘normal’’ breast epithelial cells. MCF7 was established from the pleural effusion
from a female suffering from a breast adenocarcinoma. It is classified as luminal A, ER + /PR
+/- /HER - . Using RNAseq we analysed total and polysomal (≥2n) mRNA populations in both
cell lines. Results demonstrated that in both cells polysomal transcript abundance generally
mirrored cellular transcript levels. However, in analysing transcripts from within the entire
polysomal fraction our readout lacked resolution since it could not detect movement within
polysomes (light↔heavy). Further data mining of the transcriptome revealed extensive cell-type
specific mRNA 5’leader heterogeneity within 132 genes, arising mainly due to differential TSS
(transcriptional start sites). In addition, a fraction of these differential TSSs occurred in the
absence of any significant increase in the relative transcriptional levels in each cellular context.
A number of the genes identified have already been implicated in breast cancer. The possible
implications of this heterogeneity in terms of the protein readout will be discussed.
Page 129
EMBO Conference Series: Protein Synthesis and Translational Control
94
Genome-wide scanning for recurrent alterations of translation factors in
glioblastoma multiforme
Erik Dassi, Angelika Modelska, Alessandro Quattrone
University of Trento, Italy
Presenter: Angelika Modelska
Glioblastoma multiforme (GBM) is a central nervous system tumor believed to originate from
progenitor cells in the subventricular zone (SVZ). Most often GBM develops in late adulthood
and accounts for the majority of primary brain tumors in adults. Despite intensive research, no
effective treatment is available: temozolomide, the best drug on the market now, only slightly
prolongs the 14 months median survival. Translation is the gene expression step closer to the
cell phenotype. Understanding how this process is dysregulated in cancer and determining the
key factors involved may lead to the identification of targetable regulatory mechanisms, thus
opening the way for innovative treatments of GBM. In order to study translational machinery
alterations in GBM cells, we obtained copy-number and mRNA expression data for 372 GBM
and matched-normal samples from The Cancer Genome Atlas (TCGA). These two datasets
were integrated to obtain a list of translation factors and co-factors, as annotated by Gene
Ontology, which are commonly altered in this tumor type. The analysis yielded 11 genes
showing coordinated changes in these two datasets. Amongst these, three genes resulted in a
copy-number gain and overexpression, and eight were deleted and underexpressed. Four
genes showing most extensive alterations from the above list were selected for functional
validation in glioblastoma in vitro models, including glioblastoma initiating cells (GICs). The work
includes copy-number analysis as well as determination of mRNA and protein levels. Suitable
knock-down and over-expression models are used to perform functional assays such as
proliferation, cell cycle, migration and invasion. Our first results show promising insights
allowing for better understanding of recurrent translation-linked alterations in GBM. Future
work will focus on further functional as well as molecular characterization of the downstream
effects of these events, which may lead to development of new therapies.
Page 130
Poster Abstracts
95
Investigating the mechanism of eIF3e-regulated epithelial-to-mesenchymal
transition
Guillaume Desnoyers, Laura Gillis, Stephen M. Lewis
Atlantic Cancer Research Institute, Canada
Presenter: Guillaume Desnoyers
Epithelial-to-mesenchymal transition (EMT) is an important process in cancer metastasis,
especially during breast cancer as it allows cells to adopt migratory and invasive properties that
lead to the dissemination of tumor cells throughout the body. Recently, we have uncovered an
important role for eIF3e in this process as we have shown that the educed expression of eIF3e
(which is found in up to 37% of breast cancers) leads to EMT. The eIF3e protein is a
component of the multi-subunit eIF3 complex, which binds directly to the 40S ribosome to
facilitate ribosome recruitment to mRNA and hence protein synthesis. The goal of this project
is to determine by which mechanism does a decrease in eIF3e level leads to EMT. My data
show that eIF3e-mediated EMT is at least partially achieved through the overproduction of
TGF-β, a cytokine that is known to induce EMT. More specifically, I show that mammary
epithelial cells (MCF10A) that have reduced eIF3e levels produce and excrete more TGF-β
while treatment of these cells with a specific inhibitor of the TGF-β pathway reverses EMT. In
these cells, my data show that while TGF-β production is increased, the actual tgfb1 mRNA
levels are not altered, suggesting a post-transcriptional mechanism. Furthermore, polysome
fractionation shows that the tgfb1 mRNA translation is increased in eIF3e- cells, even though
general cap-dependent translation is compromised. Interestingly, the 5' untranslated region of
the tgfb1 mRNA is unusually long and has a high GC content, two hallmarks of sequences that
contain internal-ribosome-entry-sites (IRES). Because of the role of e IF3e in bridging the
cap-binding complex (eIF4F) to the 40S ribosomal subunit, we believe that the tgfb1 mRNA
may be translated by a cap-independent mechanism that would be favored when eIF3e levels
are reduced. Experiments are underway to test this hypothesis.
Page 131
EMBO Conference Series: Protein Synthesis and Translational Control
96
Interaction between the SRP receptor and the translocon SecYEG at tme
membrane
Albena Draycheva, Thomas Bornemann, Wolfgang Wintermeyer
MPI for Biophysical Chemistry, Germany
Presenter: Albena Draycheva
Polytopic membrane proteins are inserted into the bacterial plasma membrane via the
cotranslational SRP pathway. One of the least understood steps of this process is the
interaction between FtsY and the SecYEG translocon. This interaction is crucial for the transfer
of the translating ribosome onto the translocon. Recent publications have shown that FtsY
binds to the membrane and also interacts directly with the translocon. However, only little is
known about the dynamics of the FtsY-SecYEG interaction, i.e. affinities and complex stability
during the targeting process. To address this question we have established a FRET system
between SecYEG and FtsY in order to investigate the thermodynamic and kinetic parameters
of this interaction and how it may change during the transfer of ribosomes onto the translocon.
Our data show that FtsY binds to the translocon in a nucleotide independent manner with
nanomolar affinities which are in the same range as the affinities of SRP binding to ribosomes
and of FtsY binding to SRP. The SecYEG-FtsY complex exists with a relatively long half-life of
several, supporting models that assume FtsY to be mainly membrane-associated and also
bound to the translocon. How the dynamics of this interaction is changing in the presence of
translating ribosomes is currently investigated.
Page 132
Poster Abstracts
97
Ribosomal protein S1 unfolds structured mRNAs on the ribosome for
translation initiation in Escherichia coli
Mélodie Duval1, Alexey Korepanov2, Olivier Fuchsbauer1, Pierre Fechter1, Andrea Haller3,
Attilio Fabbretti4, Ronald Micura3, Bruno Klaholz5, Mathias Springer2, Pascale Romby1,
Stefano Marzi1
1 IBMC-CNRS, France
2 IBPC-CNRS, France
3 Institute of Organic Chemistry - Leopold Franzens University, Austria
4 Laboratory of Genetics, University of Camerino, Italy
5 Institute of Genetics and of Molecular and Cellular Biology, France
Presenter: Mélodie Duval
Regulation of translation initiation is well appropriate to adapt cell growth in response to stress
and environmental changes. Many bacterial mRNAs adopt structures in their 5’ untranslated
regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs
interact with the 30S in a two-step pathway where the docking of a folded mRNA precedes an
accommodation step. Here, we demonstrate that ribosomal protein S1 endows the 30S with
an RNA chaperone activity that is essential for the binding and the unfolding of structured
mRNAs, allowing the correct positioning of the initiation codon inside the decoding channel.
Kinetic measurements suggest that the S1-induced RNA melting is the rate-limiting step in
translation initiation of structured mRNAs. We show that the first three RNA-binding domains of
S1 are essential in vivo and are crucial and sufficient for the formation of active initiation
complexes. However, S1 is not required for all mRNAs and shows specificity with regards to
the type of regulatory elements present in the 5’ untranslated regions of mRNAs. Finally, we
show that repressor proteins fine-tune translation initiation by counterbalancing the action of
S1 on the ribosome. All in all, S1 confers dynamics to the ribosome to selectively translate
unstructured and structured mRNAs.
Page 133
EMBO Conference Series: Protein Synthesis and Translational Control
98
Dissecting the message: Transcriptional and translational profiling of mouse
CA1 hippocampal neurons
Irina Epstein1, Irena Vlatkovic1, Georgi Tuschev1, Ana Babic2, Wei Chen2, Erin Schuman1
1 Max Planck Institute for Brain Research, Germany
2 Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular
Medicine (MDC) Berlin-Buch, Germany
Presenter: Irina Epstein, Irena Vlatkovic
Local protein synthesis and degradation take place in hippocampal CA1 dendrites and are the
basis for cell biological processes that modify synaptic function. In rat, more than 2500 mRNAs
have been shown to populate dendrites and/or axons in the synaptic neuropil (Cajigas et al.,
2012) revealing a high diversity of dendritic mRNAs that was not previously appreciated. Here
we take advantage of a transgenic mouse model system expressing a Bacterial Artificial
Chromosome under a promoter that drives expression of EGFP-Rpl10a fusion protein in CA1
hippocampal neurons (Gong, 2003; Heiman et al, 2008) to address questions about the
transcription and translation of mRNAs in pure population of neurons. We microdissected the
somata and neuropil layers from area CA1 of the rat hippocampus, enabling us to directly
examine the local dendritic RNAs and the fraction of localized mRNA undergoing translation.
We characterized the transgenic mice using immunocytochemistry, qRT-PCRs and Western
blot and showed the presence of EGFP in both somata and dendrites. We performed TRAP
(Translating Ribosome Affinity Purification) and isolated mRNAs that are translated from
somata and neuropil of hippocampal neurons that will be subjected to RNA-sequencing. In
parallel, we RNA-sequenced ribosomally depleted total RNA from somata and neuropil of
these mice and are presently analyzing expression of both protein coding and long non-coding
genes. By manipulating the system with pharmacological treatments we will monitor how the
transcriptome and translatome may change upon synaptic plasticity. Our study will provide
deeper understanding of the dendritic ncRNA and mRNA population as well as reveal the
global potential of local translation in hippocampal neurons.
Page 134
Poster Abstracts
99
Assaying plant ribosomes with asymmetric flow field-flow fractionation
Katri Eskelin, Leena Pitkänen, Päivi Tuomainen, Kristiina Mäkinen
University of Helsinki, Finland
Presenter: Katri Eskelin
The distribution of mRNAs among polysomes and monosomes are indicative of their
translational activity: translationally active mRNAs are associated with polysomes. Internal and
external stimuli affect the translatability of mRNAs. Therefore, changes in the ratio of free
subunits, monosomes an polysomes are informative about the physiological state of the cell.
Our aim was to study the suitability of asymmetric flow field-flow fractionation(AsFlFFF) for
ribosome purification and profiling. In AsFlFFF, the separation takes place in an open channel.
Thus, elution in gentle and keeps the analyzed molecules in native conformation. For AsFlFFF,
crude preparations of ribosomes were prepared from Nicotiana benthamiana plants using
ultracentrifugation. The injection/focusing and elution steps of AsFlFFF were optimized. Free
RNA and free protein that co-sedimented with ribosomes during ultracentrifugation were nicely
separated from the main peak. The main peak was broad and had several sub-peaks.
Treatment of the input sample with RNAseA and EDTA verified that the main peak contained
the ribosomal subunits and intact ribosomes. Finally, we collected fractions from AsFlFFF and
analyzed their protein and RNA content. Analysis of ribosomal RNA verified the position of the
subunits and ribosomes in the fractograms. Furthermore, RT-PCR analysis of the main peak
for several genes showed that ribosomes remained intact and bound to mRNAs during the
AsFlFFF analysis. Analysis of the protein content correlated with the RNA data: the protein
profiles of the main peak differed from the peak representing the free protein and free RNA. In
conclusion, our results show that AsFlFFF is well suited to study ribosome composition as well
as changes in the ratios of subunits, monosomes and poysomes during different growth
conditions of plants.
Page 135
EMBO Conference Series: Protein Synthesis and Translational Control
100
Nucleolar proteins MINA53 and NO66 are ribosomal protein histidinyl
hydroxylases
Tianshu Feng, Wei Ge, Adam Zayer, Rok Sekirnik, Nicolas Granatino, Ming Yang, Atsushi
Yamamoto, Benedikt Kessler, Peter Ratcliffe, Christopher J. Schofield, Mathew Coleman
University of Oxford, United Kingdom
Presenter: Wei Ge
Ribosomal proteins are subject to an array of post-translational modifications including
phosphorylation, ubiquitylation, and methylation. Hydroxylation is an emerging modification
catalysed by a family of oxygenases that require molecular oxygen, Fe(II), and the Krebs cycle
intermediate 2-oxoglutarate (2OG) for activity. Although 2OG-oxygenases are implicated in
diverse aspects of gene expression, their role in protein synthesis is unclear. We discovered
that two related but non-redundant 2OG-oxygenases hydroxylate proteins of the large
ribosomal subunit. MINA53 hydroxylates H39 of RPL27a, and NO66 hydroxylates H216 of
RPL8. Mass spectrometry analyses indicate that these modifications are highly abundant
(>95%), ubiquitous, and oxygen-dependent. Our work raises the intriguing possibility that the
protein synthesis machinery is targeted by enzymes that interface with metabolism and nutrient
availability.
Page 136
Poster Abstracts
101
Towards the structural characterization of co-translational action of N-terminal
methionine excision pathway enzymes
Sonia Fieulaine, Renata Grzela, Julien Nusbaum, Pierre Legrand, Beatriz Guimarães, Andy
Thompson, Alexandre Pozza, Joséphine Lai Kee Him, Patrick Bron, Thierry Meinnel,
Carmela Giglione
Centre National de la Recherche Scientifique (CNRS), France
Presenter: Sonia Fieulaine
Our lab is interested in co- and post-translational modifications of newly synthesized proteins,
especially maturation processes of their N-termini. Particularly, we investigate relations
between ribosomes and factors responsible for these essential modifications. With a
multidisciplinary approach, from in vitro to in vivo techniques, we try to understand the
complex rules that regulate the sequential or simultaneous actions of a high number of factors,
at a crucial moment for new nascent polypeptides when they emerge from exit ribosomal
tunnel. As specialist of this maturation process, our lab is currently focused on the N-terminal
methionine excision pathway (NME), which leads to the early removal of the initiator methionine
(in eukaryotic cytoplasm) or N-Formyl-methionine (in bacterial cytoplasm, mitochondria and
chloroplasts). This pathway is ensured by the sequential action of peptide deformylase (PDF)
which removes the Formyl group if present and methionine aminopeptidase (MetAP) which
then cleaves the free N-ter methionine. Here, we will present the strategy we have chosen to
solve structures of macromolecular complexes, composed of several PDFs and/or MetAPs
with specific bacterial ribosomes.
Page 137
EMBO Conference Series: Protein Synthesis and Translational Control
102
The ratio of eIF4G isoforms influences the balance between translation,
storage and degradation of mRNAs in Saccharomyces cerevisiae
Helena Firczuk, Xiang Meng, John Duncan, Oliver Sinfield, John McCarthy
University of Warwick, United Kingdom
Presenter: Xiang Meng
The eukaryotic initiation factor 4G (eIF4G) is an essential protein involved in the
5’cap-dependent initiation pathway. It is thought to associate with both ends of an mRNA and
to recruit the 43S pre-initiation complex. It has been proposed to fulfil a ‘scaffolding’ role since
it has binding sites for multiple other factors, including eIF4E, eIF4A, Pab1, eIF1 and Ded1. In
S. cerevisiae, there are two eIF4G isoforms encoded by duplicated genes TIF4631 and
TIF4632 that are ~50% identical in sequence. However, the existence of two isoforms raises
the question whether the two species have somewhat distinct functions. Although a large
functional overlap has been identified for the two isoforms, depletion of eIF4G1 reduces growth
rate by ~25 % whereas deletion of eIF4G2 results in near-wild-type characteristics. We have
performed growth competition experiments to characterise the respective contributions of the
two isoforms more precisely. In addition, each of the eIF4G genes was placed under control of
the tetO7 regulable promoter and the expression levels were titrated progressively to explore
their control effects on global translation regulation. Moreover, we observe a synthetic growth
effect for eIF4G1 and eIF4G2 deletions together with an eIF4A deletion (either TIF1 or TIF2 ),
especially severe in the case of TIF1 allele. This negative genetic interaction suggests one
possible explanation for the existence of two identical duplicated genes encoding eIF4A.
Through binding Ded1, eIF4G1 is also thought to be engaged in formation of a
Ded1-eIF4F-mRNP complex that is suppressed for translation and is compartmentalised into
stress granules. To investigate the link between translation rate and mRNA degradation,
fluorescent labelling techniques were used to track the distribution of P-bodies, stress
granules, and mRNA molecules under a range of conditions.
Page 138
Poster Abstracts
103
Spatial and Temporal Translational Control of Germ Cell mRNAs by an eIF4E
Isoform, IFE-1
Andrew Friday, J. Kaitlin Morrison, Melissa Henderson, Brett Keiper
Brody School of Medicine at East Carolina University, United States of America
Presenter: Andrew Friday
Gamete development is governed largely by regulated translation initiation on stored mRNAs.
The rate limiting step is their derepression and recruitment by initiation factors (eIF’s) to
ribosomes. eIF4E isoforms are the first factors to interact with mRNAs, specifically recognizing
their methyl guanosine cap. The nematode, C. elegans, expresses five isoforms of eIF4E
(IFE-1-5). Previously we demonstrated that three isoforms recruit unique subsets of mRNAs.
Consequently, individual IFE gene knockouts result in unique phenotypes in the soma and/or
germ line. Loss of IFE-1 causes temperature-sensitive sterility due to defective cytokinesis in
secondary spermatocytes as well as diminished oogenesis. In order to identify specific
mRNAs recruited by IFE-1 that play roles in oocyte and spermatocyte development, we
undertook a differential polysome microarray analyses called “translational state array assays”
(TSAA) on wildtype and ife-1 null mutants. We identified a unique set of mRNAs (~80)
recruited by IFE-1 for translation initiation. Among the IFE-1-regulated mRNAs are several that
are integrally involved in stages of germ cell differentiation such as the transition from mitosis to
meiosis and late stage oocyte maturation. Analysis of these mRNAs (e.g. gld-1, ran-1, vab-1,
vpr-1, nos-3 ) showed decreased translational efficiency in the absence of IFE-1,
demonstrating their specific reliance on this cap-binding isoform. Transgenic 3’UTR reporters
showed selectivity in spatial and temporal derepression and recruitment of several of these
mRNAs within individual developing germ cells in vivo. We will characterize unique proteins
and mRNAs in mRNP complexes specific to IFE-1. Our data provide evidence for spatial and
temporal regulation of unique mRNA populations by the translation initiation machinery. An
eIF4E isoform, IFE-1, executes positive, selective translational control that drives germ cell
differentiation.
Page 139
EMBO Conference Series: Protein Synthesis and Translational Control
104
Genome-wide search for novel human uORFs and N-terminal protein
extensions using ribosomal footprinting
Claudia Fritsch1, Alexander Herrmann2, Michael Nothnagel3, Karol Szafranski4, Klaus Huse4,
Frank Schumann2, Stefan Schreiber2, Matthias Platzer4, Michael Krawczak3, Jochen
Hampe2, Mario Brosch2
1 University Hospital Kiel, Germany
2 Department of Internal Medicine, University Hospital Kiel, Germany
3 Institute for Medical Informatics and Statistics, University Hospital Kiel, Germany
4 Genome Analysis Group, Fritz Lipmann Institute for Age Research, Germany
Presenter: Claudia Fritsch
So far, the annotation of translation initiation sites (TISs) has been based mostly upon
bioinformatics rather than experimental evidence. We adapted ribosomal footprinting to
puromycin-treated cells to generate a transcriptome-wide map of TISs in a human monocytic
cell line. A neural network was trained on the ribosomal footprints observed at previously
annotated AUG translation initiation codons (TICs), and used for the ab initio prediction of TISs
in 5062 transcripts with sufficient sequence coverage. Functional interpretation suggested
2994 novel upstream open reading frames (uORFs) in the 5´ UTR, 1406 uORFs overlapping
with the coding sequence and 546 N-terminal protein extensions. The TIS detection method
was validated on the basis of previously published alternative TISs and uORFs. Among
primates, TICs in newly annotated TISs were significantly more conserved than control codons,
both for AUGs and near-cognate codons. The transcriptome-wide map of novel candidate
TISs derived as part of the study will shed further light on the way in which human proteome
diversity is influenced by alternative translation initiation and regulation.
Page 140
Poster Abstracts
105
Two-step model of stop codon recognition by eukaryotic release factor eRF1
Ludmila Frolova1, Polina Kryuchkova1, Alexander Grishin2, Boris Eliseev1, Anna Karyagina3,
Elena Alkalaeva1
1 Engelhardt Institute of Molecular Biology, The Russian Academy of Sciences,
Russian Federation
2 Gamaleya Institute of Epidemiology and Microbiology, The Ministery of Healthcare,
Russian Federation
3 Belozersky Institute of Physico-Chemical Biology, Moscow State University,
Russian Federation
Presenter: Ludmila Frolova
Release factor eRF1 plays a key role in the termination of protein synthesis in eukaryotes. eRF1
consists of three domains (N, M and C) that perform unique roles in termination. Previous
studies of eRF1 point mutants and standard/variant code eRF1 chimeras unequivocally
demonstrated a direct involvement of the highly conserved N domain motifs (NIKS, YxCxxxF
and GTx) in stop codon recognition. In the current study, we extend this work by investigating
the role of the 41 invariant and conserved N domain residues in stop codon decoding by
human eRF1. Using a combination of the conservative and non-conservative amino acid
substitutions we measured the functional activity of more than 80 mutant eRF1s in an in vitro
reconstituted eukaryotic translation system and identified 15 amino acid residues essential for
recognition of different stop codon nucleotides. Furthermore, toe-print analyses provide
evidence of a conformational rearrangement of ribosomal complexes that occurs during
binding of eRF1 to mRNA and reflects stop codon decoding activity of eRF1. Based on our
experimental data and molecular modeling of the N domain at the ribosomal A site we propose
a two-step model of stop codon decoding in the eukaryotic ribosome.
Page 141
EMBO Conference Series: Protein Synthesis and Translational Control
106
Upstream open reading frames (uORFs) in the 5'UTR of PKCeta upregulate its
expression during stress of high cell density
Sigal Frost, Hadas Raveh-Amit, Jonathan Poller, Etta Livneh
Ben Gurion University of the Negev, Israel
Presenter: Hadas Raveh-Amit
It is generally accepted that mRNAs containing uORFs have an advantage in translation under
stress conditions. We have previously reported that PKCeta is among the limited number of
eukaryotic proteins described in literature as regulated via two conserved uORFs during stress
imposed by amino acid starvation. Using fusion constructs of each uORFs (uORF1 and
uORF2) with the luciferase (LUC) reporter gene, we demonstrate that translation is initiated at
both uORFs and is more frequent at uORF2, in correlation with the strength of its kozak
sequence. Translation of uORF2 was not affected by eliminating the translation initiation of
uORF1, suggesting that reinitiation at uORF2 is not affected by uORF1 probably due to large
intercisronic distance. Moreover, we show that reinitiation at the main ORF could occur after
translation of uORF2 and is not influenced by the presence of uORF1. Here we report that high
cell density also upregulates PKCeta protein expression, and that this upregulation is mediated
by its two uORFs. To examine whether the malignant state of breast cancer cells affects
translational regulation by uORFs we have examine PKCeta expression using the
non-malignant breast cells MCF10A, the non-metastatic MCF7 and the highly metastatic
MDA-MB-231 cells. The protein levels of PKCeta were enhanced when cell density was
increased in MCF10A and MCF7 cells, but not in MDA-MB-231. Interestingly, we show that
both uORFs function to enhance the translation of PKCeta in MCF7 and MCF10A cells, while
in MDA-MB-231 cells, only uORF2 functions to enhance translation, whereas uORF1 is
probably constantly leaky scanned. PKCeta was previously demonstrated to phosphorylate the
tight- junction (TJ) protein, occludin, in order to maintain epithelial TJ integrity. Thus, we
suggest that the upregulation of PKCeta expression regulated via uORFs in high cell density
conditions is important for TJ maintenance, which is disrupted in MDA-MB-231 highly
malignant cells.
Page 142
Poster Abstracts
107
Eukaryotic Initiation Factor 4AII is under MyoD control during myogenesis
Gabriela Galicia Vazquez, Sergio DiMarco, Imed Gallouzi, Jerry Pelletier
McGill University, Canada
Presenter: Gabriela Galicia Vazquez
Translational control is thought to be a key feature of muscle differentiation. Protein synthesis
rates increase within the first 24h of differentiation, and this correlates with an increase in
4EBP1 and eIF4E phosphorylation (Willett, et al. 2009). As well, an increase in the association
of PABP, eIF4G, and eIF4A with eIF4E can be observed (Willett, et al. 2009). The mTOR
inhibitor, RAD001, is capable of blocking muscle differentiation. These results suggest a role
for translational control in myogenesis. The helicase activity of eIF4F is imparted by eIF4A. In
mammals, there are two isoforms: eIF4AI and eIF4AII. These are ~90% identical at the amino
acid level, exhibit similar activity, and are interchangeable in vitro. Recently, we demonstrated
that these two isoforms are not equivalent in cells, and that the eIF4AII isoform is unable to
rescue translation under conditions when eIF4AI is suppressed (Galicia-Vazquez, et al. 2012).
To assess for differences in activity between eIF4A isoforms, we have characterized their
expression during muscle cell differentiation using the C2C12 myoblast cell line model. We
found that eIF4AI levels decrease, while eIF4AII levels increase, during the differentiation
process. This effect was observed at protein and mRNA levels. Transcriptional activation of
eIF4AII during myogenesis was confirmed using nuclear run-on assays. Analysis of the eIF4AII
promoter revealed that it harbors three MyoD binding sites and we hypothesized that eIF4AII is
controlled
by
the
master
regulator
MyoD
during
muscle
differentiation.
Chromatin-Immunoprecipitation (ChIP) analysis revealed that MyoD binds to the eIF4AII, but
not the eIF4AI promoter two days following induction of differentiation. Furthermore, luciferase
reporter assays showed stimulation of eIF4AII transcription upon over-expression of MyoD.
The possible role of eIF4AII in myotube maintenance will be discussed.
Page 143
EMBO Conference Series: Protein Synthesis and Translational Control
108
The role of ribosomal heterogeneity in translational control by metabolic
signaling.
Magdalena Gamm, Johannes Hanson
Universiteit Utrecht, The Netherlands
Presenter: Magdalena Gamm
The 79 ribosomal proteins (RP) of Arabidopsis thaliana are encoded by multigene families with
up to seven similar paralogs. Their expression is often differentially regulated by environmental
factors, but the role of this diversity remains unknown. Using RP paralog knock-out mutants,
recent studies established functional redundancy for some of the proteins, whereas a specific
function was postulated for others, based on paralog-specific phenotypes. A recent proteomic
study showed that sucrose treatment of Arabidopsis leaves leads to the enrichment of specific
ribosomal protein paralogs within the pool of actively translating ribosomes, suggesting a role
of ribosomal heterogeneity in sugar signaling. Sucrose plays a central role in plant metabolism
and acts as a signal molecule for the energy status. Energy signaling involves the conserved
kinases AMPK and TOR as central components, described to participate in the regulation of
translation. Here, sucrose and dark treatments were used as cues for the signaling pathways
to study whether changes of ribosomal composition can be correlated with alterations in
translation of A. thaliana. RNA and proteins were extracted from non-polysomal, small, and
large polysomal fractions and used for microarray and quantitative proteomic studies,
respectively. Microarray data indicates changes in translational regulation of mRNAs between
different samples. PCA shows a clear separation of data points according to the treatments,
but also according to their polysomal loading. For both conditions, clustering analysis reveals
patterns of mRNA distribution that can be attributed to specific functions. In concert, results of
a quantitative proteomic approach indicate an alteration of RP composition in the different
samples. Further studies will elucidate the link between RP composition and translational
control by energy signaling.
Page 144
Poster Abstracts
109
Molecular mechanisms of SXL-mediated translational repression
Marina García-Beyaert, Fátima Gebauer
Centre for genomic regulation (CRG), Spain
Presenter: Marina García-Beyaert
Drosophila Sex-lethal (SXL) is an RNA-binding protein that regulates gene expression at the
levels of splicing, nuclear mRNA export and translation. SXL-mediated translational repression
of msl2 mRNA, which encodes a core component of the dosage compensation complex,
ensures viability of female flies by inhibiting X-chromosome dosage compensation. SXL
orchestrates msl2 translational repression by binding to both untranslated regions (UTRs) of
the transcript. SXL bound to the 5’ UTR represses ribosomal scanning and increases the
usage of an AUG (uAUG3) upstream of the main translation initiation codon. SXL bound to the
3’ UTR inhibits ribosome recruitment by an unknown mechanism that requires binding of the
SXL co-factor UNR. We are interested in the mechanism by which SXL achieves ribosome
recruitment inhibition. We have observed that, although translation of msl2 is cap-dependent,
repression via the 3’ UTR does not require the cap structure, suggesting that SXL targets
ribosome recruitment downstream of cap-binding complex recognition. Moreover,
cap-independent repression relies on the sequence, but not the length, of the 5’ UTR. The
sequence element is different to uAUG3. We are currently performing an extensive mutational
analysis to identify the elements of msl2 5’ UTR that allow cap-independent repression. We will
also show our efforts to uncover the potential factors targeted by SXL during ribosome
recruitment.
Page 145
EMBO Conference Series: Protein Synthesis and Translational Control
110
Small molecules on the eukaryotic ribosome
Nicolas Garreau de Loubresse, Irina Prokhorova, Gulnara Yusupova, Marat Yusupov
IGBMC, France
Presenter: Irina Prokhorova
Many aspects of translation and its regulation are specific to eukaryotes, whose ribosomes are
much larger and intricate than their bacterial counterparts. Recent advances in structure
determination of the yeast 80S ribosome at high-resolution revealed the precise architecture of
eukaryote-specific elements and their interaction with the universally conserved core. It
constitutes today an experimental framework to explore the eukaryotic translation apparatus as
well as small molecules of therapeutic interest for the treatment of infectious diseases, genetic
disorders and cancers. We report the first high-resolution crystal structures of several inhibitors
bound to the 80S ribosome.
Page 146
Poster Abstracts
111
Transmembrane Protein Coxsackievirus and Adenovirus Receptor (CAR)
associates with the translation machinery and regulates protein synthesis
Songsong Geng, Patrick Fok, Josephine Nalbantoglu
McGill University, Experimental Medicine, Canada
Presenter: Songsong Geng
The Coxsackievirus and Adenovirus Receptor (CAR) is a transmembrane attachment receptor
for coxsackievirus and adenovirus. CAR is also involved in diverse biological processes such
as neuronal development and tumor suppression. Although previous studies show that CAR
promotes neurite outgrowth and inhibits in vivo brain tumor growth, little is known about the
underlying mechanisms of how CAR mediates its physiological effects. Given that the highly
conserved cytoplasmic domain of CAR may be required for these effects, our purpose is to
identify potential pathways that participate in CAR’s biological functions. To determine which
proteins interact with the cytoplasmic domain of CAR, we performed proteomic analysis which
revealed several hits with components of the protein translation machinery. We then applied
pulldown, confocal microscopy, and ribosomal centrifugation to investigate the physical
association between CAR and translational factors. We constructed CAR mutants with
truncated cytoplasmic domains to map the interaction regions between CAR and translational
proteins. We further performed in vitro and in vivo translation assays to study CAR’s functional
effects on translation. Our results demonstrate that CAR physically associates with ribosomal
proteins S6 & L4, initiation factors eIF4E & eIF4GI, elongation factor eEF1A1, and
mRNA-binding protein hnRNP U. Pulldown with CAR C-terminal truncates shows that only the
full-length cytoplasmic domain bound these factors with high affinity. We also provide evidence
that the cytoplasmic domain of CAR regulates translation both in vitro using rabbit reticulocyte
lysates, and in vivo following transfection into CHO and glioma cells. In both assays,
progressive truncation of the cytoplasmic domain reversed the observed inhibition of protein
translation. Taken together, our results suggest that CAR, a transmembrane protein,
associates with translational machinery and regulates translation.
Page 147
EMBO Conference Series: Protein Synthesis and Translational Control
112
Structural insights into Elongator function
Sebastian Glatt, Christoph W Müller
EMBL Heidelberg, Germany
Presenter: Sebastian Glatt
The eukaryotic Elongator complex consists of six highly-conserved subunits and was initially
described as a transcription elongation factor for RNA polymerase II. Although this large
molecular machine (~900 kDa) has been associated with a broad range of different cellular
activities including protein acetylation (e.g. histones, α-tubulin, Bruchpilot, …), exocytosis,
RNAi-mediated transposon suppression and zygotic paternal genome de-methylation, there is
accumulating evidence that its genuine cellular function is the specific modification of uridines
at the wobble base position of tRNAs. Notably, this singular tRNA-modifying function could
plausibly explain all of the above proposed roles of Elongator, through translational regulation
of a multitude of target mRNAs. Deciphering Elongator function is important clinically because
in certain cases of neurodegenerative diseases patients carry specific mutations in one of the
six human Elongator genes. Here, we describe the crystal structure of the Saccharomyces
cerevisiae Elp456 subcomplex, novel insights into the architecture of fully assembled
holoElongator and discuss the resulting implications for the proposed multi-functionality of
Elongator. Surprisingly, subunits Elp4, Elp5 and Elp6 share an almost identical RecA fold,
building up a hetero-hexameric ring-like structure that unexpectedly resembles hexameric
RecA-like ATPases. We show that this hexameric assembly is essential for binding to tRNAs
and analyze the regulatory role of ATP for this interaction. We provide insights into the
stoichiometry of holoElongator and novel details of its subunit communication. Our results
support a role of Elongator in translation regulation, explain the importance of each of the Elp4,
Elp5 and Elp6 subunits for complex integrity and suggest a model for the overall architecture of
the holoElongator complex.
Page 148
Poster Abstracts
113
The translational regulation of p53 and its deltaNp53 isoform using antisense
oligonucleotide strategy in vitro and in vivo
Agnieszka Gorska, Agata Swiatkowska, Mariola Dutkiewicz, Jerzy Ciesiolka
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poland
Presenter: Agnieszka Gorska
The p53 protein is one of transcription factors involved in cell cycle control, DNA repair and
apoptosis. It has been revealed that several p53 isoforms are present in the cell. One of them
is deltaNp53 isoform which binds to p53 reducing its available pool. Recently, it has been
shown that a decrease in p53 protein level by antisense oligomers may be helpful as the
additional treatment in radiotherapy of some solid malignant tumours to increase
radiosensitivity of targeted cells. In our report antisense oligonucleotide strategy was applied to
regulate translation from the initiation codons AUG1 and AUG2, for p53 and deltaNp53,
respectively. To design antisense oligomers targeting the 5' untranslated region of p53 mRNA
sites accessible to hybridization in this region were determined using libraries of 6-mer DNA
oligomers and RNase H digestion. Then, several antisense oligomers were tested in translation
system in vitro in RRL. The oligomer which binds to the lower part of the hairpin motif in which
AUG1 is embedded inhibited translation significantly. The different effect was observed in the
presence of antisense oligonucleotide which hybridizes to the hairpin U180-A218 that binds
Hdm2 protein. In this case, the level of translation from AUG2 was markedly increased. It
turned out that RNase H activity in RRL, triggered by this oligomer led to generating of
truncated transcript which was still a template for translation. Subsequently modified antisense
oligomers were examined in the cell line. In vivo results showed high potential of gapmers to
down-regulate p53 synthesis. However, for practical applications of gapmers further research
concerning their influence on cell cycle and apoptosis induction in the irradiated tumour cells
are necessary. This work was supported by the Polish Ministry of Science and Higher
Education grant no. NN301272037 to J.C. and HOMONG PLUS/2012-6/15 from Foundation
for Polish Science to AS.
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Expression and post translational modification of initiation factors determines
the response to chemotherapy in Malignant Mesothelioma.
Stefano Grosso1, Tatyana Chernova1, Xiao-Ming Sun1, Fiona Murphy1, Thomas Sbarrato1,
Jonathan Bennett2, Apostolos Nakas2, Martin Bushell1, Marion MacFarlane1, Anne Willis1
1 MRC, Toxicology Unit, United Kingdom
2 University Hospitals of Leicester, United Kingdom
Presenter: Stefano Grosso
Malignant mesothelioma (MM) is an aggressive, fatal tumour of the pleura or peritoneum,
related to asbestos exposure. To date, there is no curative therapy, the mechanism of
carcinogenesis is not well understood and there are not at present robust diagnostic markers
for the early detection of MM. To examine changes in gene expression pathways associated
with this disease, translational regulation was examined in human malignant pleural
mesothelioma (MPM) derived cell lines. First, the levels of the eukaryotic Initiation Factors (eIFs)
were assessed in MPM-derived cell lines and differences were identified in the expression of
proteins that form the eIF4F complex. Interestingly, the ratio of eIF4E/4E-BP1 expression was
critical in determining the degree of sensitivity to mTOR inhibitors. Moreover, the translation
rates were found to be independent of the presence of growth factors, suggesting an alteration
of upstream signaling pathways. To assess the pathways that were activated in MPM, kinome
profiling was performed in human MPM-derived cell lines, tissues from MPM patients and
normal untransformed mesothelial cells. The data showed that there was an increase in
signalling through Src, Akt, mTOR and STAT-3 in MPM that could lead to the increase in the
downstream phosphorylation of several eIFs. To evaluate the effect of deregulated signalling
pathways on the global protein synthesis, polysome profiling was performed on four
MPM-derived cell lines and untransformed mesothelium control cells. The data suggest an
increase of polysomal association of mRNAs involved in the mitochondrial stress response and
a corresponding increase in the synthesis of these proteins. It is now important to verify
whether the alteration of signalling pathway upstream of the translational machinery and/or the
balance between eIF4E/4E-BP1 are able to modulate the response of tumour cells to
mitochondrial stress and elucidate new ways in which to treat this disease.
Page 150
Poster Abstracts
115
Characterization of different forms of peptide deformylases and their
interaction with bacterial ribosome
Renata Grzela1, Sonia Fieulaine2, Jean-Bernard Crechet3, Willy Bienvienut2, Patrick Bron4,
Julien Nusbaum2, Alexandre Pozza2, Thierry Meinnel2, Carmela Giglione2
1 Centre National de la Recherche Scientifique, France
2 CNRS ISV, France
3 CNRS ICSN-RMN, France
4 Inserm-CNRS, France
Presenter: Renata Grzela
During protein synthesis by the ribosome, nascent polypeptides are subjected to enzymatic
processing, folding by the chaperones or targeting to place of destination. In prokaryotes the
first enzymatic factor that processes nascent chains is peptide deformylase (PDF) which
removes formyl group from the N-terminal methionine of all growing polypeptides. It has been
proposed that the C-terminal helix of E coli PDF binds directly to the ribosome next to the exit
tunnel. However, the C-terminal domain cannot be considered as the major determinant of
ribosome association for PDFs. Indeed, the C-terminal helix of PDFs, belonging to the same
class of E. coli (type I PDFs), displays no conservation of length or secondary structure. It can
be shortened almost to complete deletion with no apparent effect. My previous work allowed
confirming existence of unexpected PDF proteins into most marine phages. Interestingly, these
PDFs do not contain a C-terminal extension at all with concurrent conservation in the three
motifs that carry the catalytic residues. Preliminary results demonstrate that marine viral PDFs
are active in vivo as these enzymes are able to complement E. coli cells featuring conditional
PDF-deficiency when grown under non-permissive conditions. Thus, these newly-identified
viral PDFs can be predicted to bind directly to the elongating nascent-chains or to the
productive ribosome via another, yet unidentified dedicated ribosome-binding domain, without
the need for a C-terminal α-helix. We present here the fully characterization of these viral PDFs
including their capacity to bind to bacterial ribosome.
Page 151
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Translation control and stress granule formation during hypoxia
Katharina Haneke, Georg Stoecklin
German Cancer Research Center, DKFZ-ZMBH Alliance, Germany
Presenter: Katharina Haneke
As part of the general stress response that protects from stress-induced damage, cells inhibit
global translation, which is accompanied by polysome disassembly and stress granule (SG)
formation. Under hypoxia cells rapidly reduce the rate of global protein synthesis but
specifically enhance translation of proteins that function in the hypoxic stress response. Protein
synthesis is primarily regulated at the step of translation initiation through inhibition of the
eIF2-GTP-tRNA Met ternary complex or the cap-binding complex. Preliminary data from our
lab suggests that besides the two classical pathways of translation inhibition, phosphorylation
of eIF2 α and inhibition of mTOR, yet unidentified signaling pathways contribute to
hypoxia-induced suppression of translation. We aim to identify new kinases and phosphatases
that function in translation arrest and stress granule assembly under hypoxia using a siRNA
screening approach and SG formation as a visual read-out. We are currently setting up the
screen, and first results will hopefully be presented at the meeting. Understanding the
mechanisms that regulate global translation during hypoxia will provide a better understanding
of how cells react to oxygen deprivation – a condition that is not only characteristic of the
tumour environment but also of ischemic tissue.
Page 152
Poster Abstracts
117
A novel mechanism of initiation mediated by the Halastavi árva RNA Virus IRES
Irina Abaeva, Tatyana Pestova, Christopher Hellen
SUNY Downstate Medical Center, United States of America
Presenter: Christopher Hellen
Analysis of different structural classes of viral IRESs has shown that they promote initiation by
distinct mechanisms, each based on specific interactions with components of the translation
apparatus. Thus dicistrovirus IGR IRESs (~200nt-long) bind directly to 40S subunits, the
~350nt-long Hepatitis C virus IRES interacts independently with eIF3 and the 40S subunit, and
picornavirus IRESs (~450nt-long) bind specifically to eIF4G/eIF4A. We report here that
Halastavi árva RNA Virus (HalV), which was discovered in the intestinal content of freshwater
carp, contains a novel IRES in its genome. The structure of the 827nt-long HalV 5’UTR
(determined by probing and bioinformatic analyses) and its sequence are unrelated to those of
known IRESs. Translation of dicistronic mRNAs showed that this 5’UTR functions as an IRES
in mammalian, insect and plant cell-free extracts. IRES function is impaired by deletion of the
3’-terminal ~200nt of the 5’UTR. Factor-independent binding of 40S subunits to the IRES was
strongly stabilized by eIF3, leading to the establishment of contacts between 40S/eIF3 and the
IRES at nt. 522-3, nt. 722-5 and around the initiation codon AUG 828 . In vitro reconstitution
showed that further addition of the eIF2-TC was sufficient for 48S complex formation. eIFs
4A/4B/4G were not essential for initiation, but prevented ribosomal arrest at aberrant sites on
the IRES. eIF1/eIF1A increased the efficiency and fidelity of 48S complex formation.
Near-cognate UUG, GUG and AUU codons introduced to replace AUG 828 did not support
48S complex formation. However an AUG placed at nt. 810 was utilized in the absence of
AUG 828 , although AUG 828 was preferred when both are present. These observations
suggest a mechanism in which binding of 40S subunits/eIF3 to the IRES can lead either to
direct loading of the initiation codon region into the mRNA-binding cleft or of entry into it of
upstream sequences, in which case initiation requires subsequent scanning to AUG 828.
Page 153
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Insight into translation enhancers using ribosome profiling in Pichia pastoris
Frank Nelissen, Aafke van Vugt-Jonker, Hans Heus
Radboud University Nijmegen, The Netherlands
Presenter: Hans Heus
Production improvement of recombinant proteins in pharmaceutical and industrial
biotechnology is still a major challenge. Many proteins have been expressed using numerous
developed expression platforms, e.g., bacterial, yeast and human cell lines. However, many
recombinants suffer from poor yields and reproducibility and can vary from batch to batch.
Protein abundance in living cells seems to be primarily under translational control and in
particular the 5’untranslated regions (5’UTRs) of mRNAs can have a profound influence on
translation efficiency and protein yield. To address these issues we have applied ribosome
profiling (deep sequencing of ribosome-protected mRNA fragments) on the methylotrophic
yeast Pichia pastoris, which is widely used as a bioengineering platform for producing industrial
and pharmaceutical proteins. We have applied ribosome profiling on different strains of Pichia
pastoris, harbouring the human Interleukin 8 gene fused to 5’UTRs derived from tobacco plant
mRNAs that strongly enhance mRNA translation during pollen development. We collected
samples at various growth conditions and applied ribosome profiling to directly measure the
enhancement capacities of the 5’UTRs, determine the response of metabolic pathways and
search for other translational enhancing elements. In this presentation we highlight the
technical aspects, pitfalls, challenges, improvements and possibilities of ribosome profiling in
Pichia pastoris and discuss recent data.
Page 154
Poster Abstracts
119
Co-translational recruitment of SRP and SRP receptor to translating ribosomes
monitored in real-time
Sejeong Lee, Wolf Holtkamp, Thomas Bornemann, Albena Draycheva,
Wolfgang Wintermeyer
MPI for Biophysical Chemistry, Germany
Presenter: Sejeong Lee
Cotranslational membrane targeting of inner-membrane proteins to the protein-conducting
channel (translocon) in the plasma membrane of bacteria is conducted by the signal
recognition particle (SRP) pathway. SRP tightly binds to ribosomes synthesizing membrane
proteins and recruits the SRP receptor FtsY in a GTP-dependent manner before the ribosome
is transferred to the translocon for membrane insertion. Usually stalled ribosome-nascent-chain
complexes (RNC) were used to investigate the action of SRP and FtsY with ribosomes in
different functional states. The timing of SRP and FtsY recruitment during ongoing protein
synthesis has not been addressed so far. We have developed various FRET systems to
monitor the interaction between all components of the SRP pathway during translation in a
purified in-vitro translation system. This allowed us to monitor SRP-dependent targeting in real
time using the stopped-flow technique. We observe that SRP and FtsY are efficiently and
simultaneously recruited to ribosomes synthesizing leader peptidase (Lep), an inner membrane
protein, forming a stable targeting complex. Monitoring the SRP and FtsY interaction with
ribosomes translating Lep mRNAs of different lengths indicated that the length of the emerging
polypeptide chain influences the formation of the final targeting complex (RNC-FtsY-SRP),
despite the fact that SRP (Kd= 2 nM) and FtsY (Kd= 5 nM) bind to RNCs carrying nascent
chains of Lep with lengths between 35 and 94 amino acids with the same high affinity. Similar
experiments conducted in the presence of the translocon showed that the quaternary complex
between SRP, FtsY, translocon, and translating ribosomes is not formed, indicating that SRP
and translocon compete for the same binding site on the ribosome. The real-time FRET
approach will reveal the dynamics of the interactions of the different players of the SRP
pathway during protein synthesis.
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RNA-binding enzymes in hepatocytes
Rastislav Horos, Alfredo Castello, Bernd Fischer, Katrin Eichelbaum, Sophia Foehr,
Benedikt Beckmann, Jeroen Krijgsveld, Matthias Hentze
EMBL Heidelberg, Germany
Presenter: Rastislav Horos
The life cycle of mRNAs is determined by the spatially and temporally organized binding of
proteins (RBPs). Recent advances in the transcriptome-wide capture of RBPs revealed scores
of unexpected proteins [1,2]. Notably, many of them are metabolic enzymes, pointing to the
scarcely studied interplay between the metabolism and gene regulation [3]. As liver cells
represent a metabolically critical cell type, we determined the mRNA interactome of
hepatocytic HuH7 cell line, using in vivo RNA-protein crosslinking, isolation of mRNP
complexes by oligo-d(T) capture and analysis by quantitative mass spectrometry. We identified
726 proteins, including hundreds of previously unknown RBPs. Strikingly, the majority of
enzymes of glycolysis and TCA cycle display RNA binding activity in vivo. We also found
HSD17B10 (hydroxysteroid dehydrogenase 17 beta 10), an NAD+ dependent enzyme that
catalyzes the conversion of steroid hormones. Mutations in HSD17B10 cause a syndrome with
developmental defects, neurodegeneration and cardiomyopathy (OMIM 300438). Interestingly,
the loss of enzymatic activity of mutated forms of the enzyme does not correlate with the
severity of the phenotype. We show that HSD17B10 displays RNA-binding activity in HuH7
cells, which is reduced for the disease-associated D86G allele. Our data support the role of
moonlighting enzymes in REM (RNA/enzyme/metabolite) networks for the coordination of cell
metabolism and gene expression [3].
References:
[1] Castello, A., et al.: Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding
Proteins. Cell, 2012 [2] Baltz, A.G., et al.: The mRNA-bound proteome and its global
occupancy profile on protein-coding transcripts. Mol Cell, 2012 [3] Hentze, M.W. and T. Preiss:
The REM phase of gene regulation. Trends Biochem Sci, 2010
Page 156
Poster Abstracts
121
BOP1 mutant expression impairs ribosome biogenesis and activates mTORC1
signalling
Valentina Iadevaia1, Rui Liu1, Peter Taylor2, Ze Zhang1, Julien Averous3, Christopher Proud1
1 University of Southampton, United Kingdom
2 University of Dundee, United Kingdom
3 INRA Clermont-Theix, France
Presenter: Valentina Iadevaia
The ribosome biogenesis is one of the major cellular activities that take place in the nucleolus.
It involves the transcription and processing of pre-ribosomal RNA (pre-rRNA) and sequential
assembly of the mature rRNAs with a large number of ribosomal proteins (RPs) to form 40S
and 60S subunits. Signaling through mTORC1 (mammalian target of rapamycin complex 1)
activates the transcription and processing of rRNA and the translation of the mRNAs for RPs.
BOP1 (block of proliferation 1) is a conserved nucleolar protein involved in rRNA processing of
60S ribosome subunit. The expression of an interfering truncation mutant of BOP1 (BOP1 D)
impairs pre-rRNA processing. Here we show that that BOP1 D expression causes activation of
mTORC1 signaling. HEK293 cells expressing BOP1 D show increased phosphorylation of
mTORC1 substrates (S6K1 and 4E-BP1). Concomitantly with this, we observe stimulation of
proteins involved in translation initiation (eIF4E-eIF4G complexes) and elongation (eukaryotic
elongation factor 2; eEF2). In the latter case, BOP1 D causes both inactivation and down
regulation of eEF2 kinase, an inhibitor of translation elongation. Since mTORC1 promotes the
translation of the 5’-TOP mRNAs, which encode for RPs, we monitored the polysomal
distribution of these mRNAs (Rps). After BOP1 D induction, the 5’-TOP mRNAs (eEF1A,
RpS19 and RPL11) remained associated with the polysomes despite the overall decrease in
polysomes levels and in overall protein synthesis. The activation of mTORC1 in situations
where pre-rRNA processing is defective may represent a concerted attempt by the cell to
‘rescue’ both mRNA translation and ribosome production in situations where the latter is
deficient and ribosome subunits become depleted.
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The decameric structure of SelA reveals the bacterial selenocysteine formation
mechanisms
Yuzuru Itoh1, Markus Bröcker2, Shun-ichi Sekine3, Gifty Hammond2, Shiro Suetsugu1, Dieter
Söll2, Shigeyuki Yokoyama3
1 The University of Tokyo, Japan
2 Yale University, United States of America
3 RIKEN, Japan
Presenter: Yuzuru Itoh
Selenocysteine (Sec) is the 21st genetically encoded amino acid, and is synthesized on its
transfer RNA (tRNASec). Sec is synthesized as tRNASec-ligated form from seryl-tRNASec
(Ser-tRNASec). In Archaea and Eukaryotes, the Ser moiety of Ser-tRNASec is phosphorylated
by phosphoseryl-tRNASec kinase. Generated phosphoseryl-tRNASec (Sep-tRNASec) is
converted to selenocysteinyl-tRNASec (Sec-tRNASec) by SepSecS, depending on binding of
the Sep phosphate group without discriminating tRNASec from serine tRNA (tRNASer). In
contrast, in Bacteria, SelA directly forms Sec-tRNASec from Ser-tRNASec. Selenophosphate
is the activated selenium donor used by both SepSecS and SelA. In this study, we determined
the crystal structures of Aquifex aeolicus SelA with and without tRNASec, revealing that SelA is
a ring-shaped homodecamer (pentamer-of-dimers) that binds ten tRNASec molecules. One
tRNASec molecule interacts with two intimate dimers of SelA. The N-terminal domain of SelA
interacts with the tRNASec-specific D arm, thereby discriminating tRNASec from tRNASer. We
also determined the structure of SelA in complex with thiosulfate, an analog of
selenophosphate, indicating the catalytic mechanism of SelA. The SelA structures and in-vivo
and in-vitro assays showed that four subunits of SelA is required for the Ser to Sec conversion
of one Ser-tRNASec, i.e., subunits A and B specifically binds tRNASec and the catalytic site
located at the interface between subunits C and D converts the Ser moiety of Ser-tRNASec to
Sec. There results revealed that the decameric-ring architecture is essential for the SelA
functions. This study was reported in the journal Science on April 5, 2013.
Page 158
Poster Abstracts
123
Alu RNPs target 40S ribosomal subunits to repress translation initiation
Elena Ivanova1, Audrey Berger1, Elena Alkalaeva2, Anne Scherrer1, Katharina Strub1
1 University of Geneva, Switzerland
2 Engelhardt Insitute of Molecular Biology, Russian Federation
Presenter: Elena Ivanova
In primate cells, non-coding cytoplasmic Alu RNAs transcribed from the repetitive Alu elements
form complexes, referred to as Alu RNPs, with the protein heterodimer SRP9/14. SRP9/14 is
also a part of the signal recognition particle (SRP), a ribonucleoprotein complex, which plays a
key role in protein translocation into the endoplasmic reticulum. The SRP RNA gene is the
phylogenetic precursor of the Alu elements. We found that biochemically purified Alu RNPs
inhibit translation initiation in rabbit reticulocyte lysate on capped and non-capped mRNAs in a
dose-dependent manner whereas the protein and the RNA moieties alone had no effect. The
identity of the RNA moiety in the Alu RNP was important for function: RNPs assembled on Alu
RNAs transcribed from the youngest Alu Y elements were the most efficient in translation
inhibition indicating that this function was acquired during evolution. In the protein moiety of the
Alu RNP, the presence of a positively charged domain composed of the C-terminal
pentapeptide in SRP14 and three lysines in SRP9 was required to inhibit translation initiation.
To investigate the mechanism of inhibition, we studied the effect of Alu RNPs on the in vitro
assembly of different translation complexes. We found that Alu RNPs interfere with 48S
complex formation by inhibiting the recruitment of the mRNA to the 43S complex. Moreover,
Alu RNP repressed ribosomal complex assembly on the cricket paralysis virus IRES which
occurs independently of any initiation factors, suggesting that Alu RNPs has a direct effect on
40S subunit. Consistently, we found SRP9/14 to be bound to 40S subunits upon their
incubation with Alu RNPs. Our results describe a novel translational control mechanism in
which Alu RNA ensures functional binding of SRP9/14 to the 40S ribosome and thereby
prevents cap-dependent as well as IRES-mediated ribosome recruitment to the mRNA. This
pathway might be activated in response to viral infection to interfere with IRES-dependent
translation initiation.
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Analysis of the circadian translatome in mouse liver by ribosome profiling
Peggy Janich, Bulak Arpat, David Gatfield
University of Lausanne, Switzerland
Presenter: Peggy Janich
The circadian clock is an endogenous timekeeping mechanism that allows organisms to
anticipate and synchronize physiological and metabolic processes to the appropriate time of
the day. In mammals, circadian clocks are present in almost all tissues including the liver,
where they control the rhythmic expression of genes involved in energy homeostasis,
detoxification, fatty acid synthesis and glucose metabolism. Several genome wide
transcriptome studies revealed that up to 10% of all transcripts in mouse liver are rhythmically
expressed throughout the day. However, proteomic approaches have pointed out that
rhythmic transcription is not a prerequisite for rhythmic protein abundance, since several
rhythmic proteins are translated from non-rhythmic transcripts. Thus, the number of rhythmic
proteins per cell is expected to be considerably larger. We have used the technique ribosome
profiling, which is based on the deep-sequencing of ribosome protected mRNA fragments, to
measure protein synthesis rates on a global scale. In parallel we generated total RNA-seq data
sets from the same mouse liver samples collected every 2 hours during a period of 48 hours to
further determine the extent to which translation occurs from either rhythmic or non-rhythmic
transcripts. Together with additional analyses regarding features of translation regulation such
as the use of upstream open reading frames and alternate translation initiation sites, our study
will add another layer of complexity to circadian regulated networks and help to improve the
understanding of clock controlled signaling pathways that govern liver physiology.
Page 160
Poster Abstracts
125
Transcript profile of p21 gene and subcellular localization of the protein in
cardiac stress
Helena Kaija1, Terttu Särkioja2, Marja-Leena Kortelainen1, Heikki Huikuri3, Katja Porvari1
1 University of Oulu, Institute of Diagnostics, Department of Forensic Medicine, Finland
2 National Institute for Health and Welfare, Finland
3 University of Oulu, Institute of Clinical Medicine, Department of Internal Medicine, Finland
Presenter: Katja Porvari
Regulatory pathways are triggered in heart tissue under acute or chronic stress conditions
leading either to adaptation and remodeling or to disturbed or even fatal outcome. Acute
myocardial infraction leads to different survival strategies than chronic hypoxia-related heart
diseases. Severe accidental hypothermia causes cardiac arrest whereas cardioprotection is
achieved by controlled therapeutic hypothermia. Protein and mRNA level studies of cadaver
heart tissue samples combined with information obtained from forensic autopsy reports
enabled us to compare changes in molecular events in various antemortem conditions using
non-cardiac trauma death cases as control. Cyclin-dependent kinase inhibitor p21 (CDKN1A,
Cip1, Waf1) located in nucleus is known to inhibit cell cycle progression, but evidence in recent
years has suggested that it has an important role in promoting cell proliferation and regulating
apoptosis. We found that the mRNA expression patterns of p21 variants varied according to
the cause of death. Subcellular localization and interactions of p21 protein in cytosol in different
conditions are of major importance in order to clarify the functions of p21. In chronic hypoxia
increased expression of p21 variant alt-a and cytoplasmic localization of p21 protein in
vascular smooth muscle cells could be signs of vascular wall cell adaptation to long-term
hypoxia. Increased expressions of p53 and Pim-1, regulating expression and subcellular
localization of p21, respectively, were also detected in these cases. Cardioprotection by
hypothermia might rise from increased p21 variant V1 expression and cytoplasmic protein p21
localization in cardiac myocytes. p21 B variant mRNA expression was in our study
characteristic to acute ischemia deaths.
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Translational control by initiation factors drives germ cell fate during sperm and
oocyte differentiation
Melissa Henderson1, Andrew Friday1, Jacob Subash1, J. Kaitlin Morrison1, Vince Contreras1,
Anren Song2, Robert Rhoads2, Brett Keiper1
1 Brody School of Medicine at East Carolina University, United States of America
2 LSU Health Sciences Center, United States of America
Presenter: Brett Keiper
Germ cells rely on mRNA translational control to regulate the de novo synthesis of proteins that
drive differentiation to sperm and oocytes. mRNA repression and poly(A) elongation have been
adequately described, but the mechanisms for subsequent positive translational control
(recruitment) have been overlooked. We have begun to uncover a role of specific isoforms of
translation initiation factors (eIF4) in mRNA recruitment. Our lab described mRNA-selective
functions for individual isoforms of eIF4E (IFE-1, -2, and -3) in germ cells of the nematode C.
elegans. Germ line stem cells in hermaphrodite worms differentiate sequentially into sperm and
oocytes. Mutations in each eIF4E isoform reduce fertility, but in unique ways. Each alters a
different fate of gamete differentiation. These result in blocked maturation steps, inefficient
meiotic recombination, or even switching oogenesis to a sperm fate. We identified mRNAs that
uniquely require IFE-1 or -2 for efficient initiation using biochemical fractionation and a
translation state array analysis (TSAA) from null mutant worms. Identified mRNAs encode
proteins of critical function in oocyte and/or spermatocyte differentiation. IFE-1 is required
efficiently translation of pos-1, gld-1, mex-1, and oma-1 mRNAs. Spermatocytes lacking IFE-1
fail in the final budding/cytokinesis step, accumulating as multinucleated secondary
spermatocytes; oocytes have slowed growth and maturation. IFE-2 is required to synthesize
proteins for meiotic crossover and chromosome segregation (msh-4 and msh-5 mRNAs). The
loss of the eIF4E isoform IFE-3 causes a switch in germ line stem cell fate. Those that would
normally become oocytes differentiate instead into mature sperm. Thus, in reproductive cells,
eIF4 factors can select specific mRNAs for translation to drive acute developmental functions.
The evidence suggests specific roles for eIF4 factors in mediating translational control to
promote gamete differentiation.
Page 162
Poster Abstracts
127
Translational control of p53 and ‘little-brother’ delta N-p53 in
glucose-deprivation
Debjit Khan1, Ridhima Lal1, Aritra Das2, Arandkar Sharathchandra1, Aanchal Katoch1, Samit
Chattopadhyay2, Saumitra Das1
1 Indian Institute of Science, India
2 National Centre for Cell Science, India
Presenter: Debjit Khan
Tumor suppressor protein p53 is critical in maintaining cellular homeostasis and genomic
integrity. Earlier work in our laboratory led to the discovery of Internal Ribosome Entry Sites
(IRESs) within the p53 mRNA that regulate the translation of full-length p53 and its N-terminally
truncated isoform, delta N-p53. These IRESs are regulated under physiological and
pathological conditions by differential binding of proteins called IRES-trans acting factors
(ITAFs). Polypyrimidine-tract binding protein (PTB) and Annexin A2 are two such ITAFs that
regulate the IRES mediated translational outcome from p53 mRNA, under genotoxic and ER
stresses and at G2-M transition. Stress such as hypoxia or nutrient depletion that drive tumor
progression and maintenance partly exerts such effect by influencing IRES mediated
translational control. To understand the translational regulation of p53 mRNA, we tested its
IRES activity upon glucose-depletion in H1299 non-small cell lung carcinoma cells. Results
suggest that glucose-depletion induces p53 IRES activity as seen in bicistronic reporter
assays. Interestingly, we found Scaffold/Matrix associated region protein 1 (SMAR1), a
predominantly nuclear protein is abundant in the cytoplasm under glucose-deprivation, but
PTB does not show similar relocalization. In vitro and in cellulo binding studies revealed that
SMAR1 associates with p53 IRES sequences in H1299 and A549 cell-lines. Our results also
show that RNAi mediated knockdown of SMAR1 can decrease p53 IRES activities in normal
conditions as well as under glucose-deprivation. Using SMAR1 knockdown-mediated
reduction in delta N-p53 protein levels, we demonstrate that subsequent transcriptional
activation of the 14-3-3 sigma gene, a known target of delta N-p53, is also reduced. Thus
glucose-deprivation, a form of nutrient-depletion stress, can induce p53 IRESs through
increased cytoplasmic abundance and probably, a consequent increase in association of p53
RNA with SMAR1, a novel ITAF.
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Molecular pathways for building a secretory cell
Konstantin Khetchoumian1, Aurelio Balsalobre1, Helen Christian2, Jacques Drouin1
1 IRCM, Canada
2 University of Oxford, United Kingdom
Presenter: Konstantin Khetchoumian
The pituitary gland is an endocrine organ containing six cell lineages specialized in large-scale
hormone production. For example, proopiomelanocortin (POMC)-secreting cells increase their
hormone production about 100-fold after birth. Tpit is a POMC cell-specific Tbox transcription
factor (TF) that drives terminal differentiation of POMC lineages. While normal POMC cells
increase their size and expand organelle content after birth, Tpit-deficient cells remain small
and have few secretory organelles. We used the Tpit knockout model of blocked differentiation
to query mechanisms required for establishment of the secretory apparatus. Using expression
profiling and Tpit ChIPseq, we found that Tpit controls the gene regulatory network for
implementation of the secretory capacity (“the secretory network”) through direct regulation of
2 critical TFs, incl. the Unfolded Protein Response (UPR) regulator XBP1. Moreover, Tpit
directly targets the promoters/enhancers of downstream secretory pathway genes through
direct DNA binding or tethering with XBP1 or the other Tpit-dependent TF. Gain-of-functions
for these 2 TFs led to increased cell size, organelle, protein and RNA contents, and regulated
hormone secretion. Classically known as a cytoprotective stress response to protein misfolding
that decreases protein synthesis, the UPR has also been implicated in a so-called
“physiological UPR” that would promote secretory capacity. We have now identified a critical
TF that accounts for this function and modulates XBP1-dependent actions to support the
heavy load of protein synthesis required in secretory cells. Together, these TFs and their target
genes define the secretory network. Remarkably, we found that the tissue-specific TF Tpit
takes control of this gene network responsible for transforming POMC cells into “hormone
producing factories”.
Page 164
Poster Abstracts
129
The molecular mechanism of dendritic RNA localization in primary neurons
Michael Kiebler
LMU Munich, Germany
Presenter: Michael Kiebler
In mature hippocampal neurons, a subset of RNAs localizes to dendrites within
ribonucleoprotein particles (RNPs). We have identified a series of molecular markers that allow
us to follow dendritic mRNA transport in living neurons and to identify the underlying molecular
machinery. This includes the brain-specific double-stranded RNA-binding protein 2, Barentsz
(MLN51, Casc3), Pumilio 2 amongst others. Furthermore, we know have the first set of
physiological targets in hand that are transported in those RNPs to distal synapses. I will
present evidence that dendritic RNA localization is coupled to translational control and mRNA
stability. In conclusion, neuronal RNA granules are much more heterogeneous and dynamic
than previously anticipated.
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EMBO Conference Series: Protein Synthesis and Translational Control
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Pbp1 is involved in the Ccr4 and Khd1-mediated regulation of cell growth
through the association with ribosomal proteins, Rpl12a and Rpl12b
Yuichi Kimura, Kenji Irie
University of Tsukuba, Japan
Presenter: Yuichi Kimura
The Saccharomyces cerevisiae Pbp1 (polyA-binding protein (Pab1)-binding protein) is believed
to be involved in RNA metabolism and regulation of translation, since Pbp1 regulates a length
of poly(A) tail, and is involved in stress granule (SG) formation. However, a physiological
function of Pbp1 remains unclear since the pbp1 Δ mutation has no obvious effect on cell
growth. In this study, we show that PBP1 genetically interacts with CCR4 and KHD1 , which
encode a cytoplasmic deadenylase and an RNA binding protein, respectively. Ccr4 and Khd1
modulate a signal from Rho1 in the cell wall integrity pathway by regulating the expression of
RhoGEF and RhoGAP, and the double deletion of CCR4 and KHD1 confers severe growth
defect displaying cell lysis. We found that the pbp1 Δ mutation suppressed the growth defect
caused by the ccr4 Δ khd1 Δ mutation. The pbp1 Δ mutation also suppressed the growth
defect caused by double deletion of POP2 , encoding another cytoplasmic deadenylase, and
KHD1 . Deletion of the gene encoding previously known Pbp1-interacting factor, Lsm12,
Pbp4, or Mkt1 did not suppress the growth defect of the ccr4 Δ khd1 Δ mutation,
suggesting that Pbp1 acts independently of these factors in this process. Then we screened
novel Pbp1-interacting factors, and found that Pbp1 interacts with ribosomal proteins, Rpl12a
and Rpl12b. Similarly to the pbp1 Δ mutation, the rpl12a Δ and rpl12b Δ mutation also
suppressed the growth defect caused by the ccr4 Δ khd1 Δ mutation. Our results suggest
that Pbp1 is involved in the Ccr4 and Khd1-mediated regulation of cell growth through the
association with Rpl12a and Rpl12b.
Page 166
Poster Abstracts
131
Using the response to cold stress to analyse the role of SUMOylation in RNA
metabolism
John Knight1, Amandine Bastide1, Diego Peretti1, Joanne Roobol2, Jo Roobol3, Martin
Bushell1, Mark Smales3, Giovanna Mallucci1, Anne Willis1
1 MRC Toxicology Unit, United Kingdom
2 School of Biosciences, University of Kent, United Kingdom
3 Centre for Molecular Processing and Protein Science Group, United Kingdom
Presenter: John Knight
Exposure to sub-optimal temperatures is perhaps the most common stress encountered by
cells in a physiological context. As well as day to day cooling due to the environment,
hypothermia is used as a tool in operative medicine and to reduce costs during mammalian
expression of recombinant proteins. We, and others, culture mammalian cell lines at 32°C as a
model system to study cold stress. This moderate cold-shock is not lethal but does induce
intracellular changes, from altered nuclear gene expression to membrane lipid redistribution. At
the level of translation the global rate of protein synthesis is reduced by cold stress, but
specific transcripts are able to increase their protein abundance by increasing relative
polysomes association. SUMOylation is a post-translational peptide modification
mechanistically similar to the ubiquitin ligation system. However, SUMOylation differs from
ubiquitylation, having a distinct range of substrates, conserved motifs driving conjugation and
SUMO-protein interactions, and most notably in the molecular outcome of SUMO modification.
SUMOylation can enhance, suppress or entirely alter the function of its target protein, allowing
for rapid and reversible modulation of function. Our interest comes from the observation that
SUMOylation is upregulated in cell and animal models of cold stress, forming high molecular
weight conjugates that are also common to other stresses. Recent analyses of the
SUMOylated proteome have revealed enrichment for proteins involved in RNA metabolism.
SUMO modified proteins are involved in RNA synthesis, splicing, modification, localisation,
translation and degradation. We aim to use cold stress as a tool to analyse the alterations in
global RNA metabolism attributable to protein SUMOylation. This may have wider applicability
to alternative cell stresses and assist the medical and industrial use of hypothermia.
Page 167
EMBO Conference Series: Protein Synthesis and Translational Control
132
Dynamics of selenocysteine incorporation in bacterial translation
Suresh Kotini, Frank Peske, Marina Rodnina
MPI for Biophysical Chemistry, Germany
Presenter: Suresh Kotini
In bacteria, Selenocysteine (Sec) incorporation into proteins requires the function of four gene
products, SelA, SelB, SelC & SelD. A stop codon, UGA, which is usually recognized by the
release factor 2 (RF2), is recoded into Sec when a SECIS is present immediately downstream
of the UGA codon. The mechanism of UGA decoding by Sec-tRNA Sec on the ribosome
remains unclear, largely because detailed mechanistic studies were so far hampered by the
lack of reliable in vivo and in vitro assays. The efficiency of Sec incorporation into E. coli
proteins in vivo was reported to be low and depend on the growth stage, raising the possibility
that Sec incorporation is under translational control by auxiliary factors unknown so far or by
changes in the concentration of RF2. To test this suggestion, we constructed a dual reporter
system and validated its performance using Western blots and luciferase assays. In contrast to
previous reports, which suggested a very low (3-5% efficiency of Sec incorporation) in rapidly
growing cells, we found a robust 40% of Sec insertion. Furthermore, in contrast to reports
suggesting an increase of Sec incorporation in slow growing cells, the expression did not
depend on the growth conditions. Overexpression of RF2 repressed synthesis of
selenoproteins, indicating that RF2 competes with SelB•GTP•tRNA Sec for the UGA codon;
however, the extent of inhibition is remarkably mild given the large excess of RF2 over SelB
and tRNA Sec in the cell. To study the competition between RF2 and the Sec machinery, we
designed an in vitro translation system for Sec incorporation which would authentically reflect
the in vivo speed and efficiency of selenoprotein synthesis using a fragment of natural
selenoprotein FDH H. We describe the first example of the fully reconstituted, high-yield in vitro
translation system designed to study the mechanism of selenoprotein synthesis.
Page 168
Poster Abstracts
133
Investigating the consequences of eIF4E2 (4EHP) interaction with
4E-Transporter on its cellular distribution in HeLa cells
Dorota Kubacka1, Anastasiia Kamenska2, Helen Broomhead2, Nicola Minshall2, Edward
Darzynkiewicz1, Nancy Standart2
1 University of Warsaw, Poland
2 University of Cambridge, United Kingdom
Presenter: Dorota Kubacka
In addition to the canonical eIF4E cap-binding protein, eukaryotes have evolved sequence–
related variants with distinct features, some of which have been shown to negatively regulate
translation of particular mRNAs, but which remain poorly characterised. Mammalian eIF4E
proteins have been divided into three classes, with class I representing the canonical
cap-binding protein eIF4E1. eIF4E1 binds eIF4G to initiate translation, and other eIF4E-binding
proteins such as 4E-BPs and 4E-T prevent this interaction by binding eIF4E1 with the same
consensus sequence YX 4 Lφ. We investigate here the interaction of human eIF4E2 (4EHP), a
class II eIF4E protein, which binds the cap weakly, with eIF4E –transporter protein, 4E-T. We
first show that eIF4E1/2:4E-T ratios range from 3:1 to 50:1 in mammalian cell lines and provide
evidence that eIF4E2 binds 4E-T in the yeast two hybrid assay, as well as in pull-down assays
and by recruitment to P-bodies in mammalian cells. We also show that while both eIF4E1 and
eIF4E2 bind 4E-T via the canonical YX 4 Lφ sequence, nearby downstream sequences also
influence eIF4E:4E-T interactions. Indirect immunofluorescence was used to demonstrate that
eIF4E2, normally homogeneously localised in the cytoplasm, does not redistribute to stress
granules in arsenite-treated cells, nor to P-bodies in Actinomycin D-treated cells, in contrast to
eIF4E1. Moreover, eIF4E2 shuttles through nuclei in a Crm1-dependent manner, but in an
4E-T–independent manner, also unlike eIF4E1. Altogether we conclude that while both
cap-binding proteins interact with 4E-T, and can be recruited by 4E-T to P-bodies, eIF4E2
functions are likely to be distinct from that of eIF4E1, both in the cytoplasm and nucleus,
further extending our understanding of mammalian class I and II cap-binding proteins.
Page 169
EMBO Conference Series: Protein Synthesis and Translational Control
134
Orthogonal Biosystems: Cell-free synthesis of posttranslationally modified
membrane proteins
Robert Quast, Andreas Brödel, Srujan Dondapati, Christian Hoffmeister, Devid Mrusek, Rita
Sachse, Marlitt Stech, Stefan Kubick
Fraunhofer Institute for Biomedical Engineering (IBMT), Germany
Presenter: Stefan Kubick
Various genome sequencing projects have greatly accelerated the discovery of novel genes
encoding membrane proteins. In contrast, the molecular analysis of membrane proteins lags
far behind that of cytosolic proteins. Preparing high quality samples of functionally folded
proteins represents a major bottleneck that restricts further structural and functional studies.
Cell-free protein expression systems, in particular those of eukaryotic origin, have recently
been developed as promising tools for the rapid and efficient production of a wide variety of
membrane proteins. A large number of these proteins, however, require posttranslational
modifications for optimum function. Several membrane proteins have been expressed in vivo
to date, most of them being functionally, antigenically, and immunogenically similar to their
authentic counterparts. This is mainly due to the properties of cultured eukaryotic cells, which
are able to carry out many types of posttranslational modifications. Based on these versatile
properties of cultured cell lines, we have developed a technique for the standardized
production of translationally active eukaryotic lysates from insect cells. Our homogenization
procedure avoids any serious breakdown of membrane vesicles already existing in the
cytoplasm of the prepared eukaryotic cells. We have demonstrated the functional integrity of
these subcellular components by showing signal peptide cleavage as well as glycosylation of
cell-free expressed membrane proteins. Moreover, we have expanded our cell-free protein
expression system by the insertion of orthogonal tRNA/synthetase pairs to facilitate the
cotranslational and site directed incorporation of non-canonical building blocks. These
fluorescently labeled and chemoselective moieties enable the site-specific modification of de
novo synthesized membrane proteins.
Page 170
Poster Abstracts
135
Modifying the ppGpp scaffold: a molecular toolkit for investigation of the
stringent response
Pavel Kudrin1, Jelena Beljantseva1, Vallo Varik1, Tanel Tenson1, Dominik Rejman2, Vasili
Hauryliuk1
1 University of Tartu, Institute of Technology, Estonia
2 Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Czech Republic
Presenter: Pavel Kudrin
The stringent response is a central bacterial regulatory pathway mediated by the alarmone
nucleotide ppGpp1. Its cellular level is controlled by the RelA-SpoT Homologue enzymes,
which either synthesize or degrade ppGpp in response to various stress stimuli. ppGpp exerts
its regulatory role by modulating the activity of numerous enzymes2: RNA polymerase,
translational GTPases, DnaG primase, and, as we have demonstrated recently, activating the
ppGpp-synthetic activity of RelA itself3. Since the stringent response regulates bacterial
virulence and antibiotic tolerance, development of specific inhibitors is a promising strategy for
the design of novel antibacterials. Several ppGpp-based inhibitors were described but in the
current form their efficiency is not sufficient for entering the pharmaceutical pipeline4. We have
tested in an in vitro stringent response system a panel of compounds based on the ppGpp
scaffold in order to (1) characterize the molecular determinants of ppGpp-mediated RelA
activation, and (2) to develop efficient and specific ppGpp-based stringent response inhibitors
with good bioavailability properties.
References:
[1] Atkinson, G. & Hauryliuk, V. Evolution and Function of the RelA/SpoT Homologue (RSH)
Proteins. In: eLS. John Wiley & Sons, Ltd: Chichester (2012). [2] Dalebroux, Z. D. & Swanson,
M. S. Nat Rev Microbiol 10, 203-212, (2012). [3] Shyp, V. et al. EMBO Rep 13, 835-839,
(2012). [4] Wexselblatt, E. et al. PLoS Pathog 8, e1002925, (2012).
Page 171
EMBO Conference Series: Protein Synthesis and Translational Control
136
Purification and crystallization of the C-terminal domain of eukaryotic
translation initiation factor 5 (eIF5) in complex with an N-terminal fragment of
eIF2ß
Ralf Ficner, Bernhard Kuhle
Georg-August University Göttingen, Germany
Presenter: Bernhard Kuhle
Interactions between the eukaryotic translation initiation factors 2 (eIF2) and 5 (eIF5) play a
critical role at various steps during translation initiation. It has been shown that the main
interaction occurs between a region in the C-terminal domain of eIF5 rich in aromatic and
acidic side chains and three lysine-patches located in the N-terminal domain of the β-subunit
of eIF2 [1-3] . Besides a mere role in the recruitment of eIF5 to eIF2 these interactions seem to
be important for Multi Factor Complex (MFC) assembly and scanning [4] and were recently
proposed to play a direct role in start codon recognition [5] . In order to gain structural insight
into the mode of these interactions, a complex comprising the C-terminal domain of yeast eIF5
and a fragment of eIF2β(NTD) containing two of the three lysine-patches was purified and
crystallized. The structure was finally solved at a resolution of 2.0 Å.
References:
[1] Das S, Maiti T, Das K, et al. Specific Interaction of Eukaryotic Translation Initiation Factor 5
(eIF5) with the β-Subunit of eIF2. J Biol Chem, 1997, 272 (50):31712–31718 [2] Asano K,
Krishnamoorthy T, Phan L, et al. Conserved bipartite motifs in yeast eIF5 and eIF2Be,
GTPase-activating and GDP–GTP exchange factors in translation initiation, mediate binding to
their common substrate eIF2. EMBO J, 1999, 18 (6):1673–1688 [3] Das S, and Maitra U
Mutational Analysis of Mammalian Translation Initiation Factor 5 (eIF5): Role of Interaction
between the β Subunit of eIF2 and eIF5 in eIF5 Function In Vitro and In Vivo. Mol Cell Biol,
2000, 20 (11):3942–3950 [4] Asano K, Shalev A, Phan L, et al. Multiple roles for the
C-terminal domain of eIF5 in translation initiation complex assembly and GTPase activation.
EMBO J, 2001, 20 (9):2326–2337. [5] Luna RE, Arthanari H, Hiraishi H, et al. The C-Terminal
Domain of Eukaryotic Initiation Factor 5 Promotes Start Codon Recognition by Its Dynamic
Interplay with eIF1 and eIF2β. Cell Reports, 2012, 1:689–702.
Page 172
Poster Abstracts
137
Analysis of human Argonaute 1 5’ untranslated region shows internal ribosome
entry site activity
Rafaela Lacerda, Alexandre Teixeira, Ana Marques-Ramos, Luísa Romão
Instituto Nacional de Saúde Doutor Ricardo Jorge, Portugal
Presenter: Rafaela Lacerda
Argonaute (AGO) proteins are present in all described RNA-induced silencing complexes
(RISC) as the catalytic components responsible for RNA interference (RNAi). In addition, these
proteins are thought to have regulatory functions in stem cell self-renewal, including cancer
stem cells, and translation regulation through the RNA-mediated gene silencing mechanism as
a component of RISC. In human cells there are eight AGO-like proteins grouped in two
families: the eIF2C/AGO subfamily and the PIWI subfamily. AGO1, a member of the first
subfamily, is encoded by the human eIF2C1 gene. This gene is ubiquitously expressed at low
to medium levels and it is highly conserved during evolution reflecting its important
physiological role. Recent studies concluded that eIF2C1 protein in overexpressed in colorectal
cancer relative to adjacent non-cancer tissue. This evidence lead us to suspect that
translational levels of AGO1 are sustained due to the presence of an internal ribosome entry
site (IRES) in the 5’ untranslated region (UTR) of the transcript encoding this protein. IRESs are
structures that can directly recruit ribosomes to the vicinity of the main AUG, thus allowing
translation initiation to occur in a cap-independent way. To confirm this hypothesis, we
transfected colorectal cancer HCT116 cells and cervical cancer HeLa cells with an AGO1
5’UTR-containing dicistronic vector and observed a 2-fold increase in luciferase activity in both
cell lines, when compared to the cells transfected with the empty counterpart ( P <0.05).
Transfection of the corresponding promoterless plasmids ruled out the hypothesis of this fold
to be due to the existence of a cryptic promoter in the DNA encoding the UTR. In addition,
RT-PCR analysis of the dicistronic mRNAs confirmed that no cryptic splicing has occurred. All
these data suggest the presence of an IRES in the AGO1 5’UTR. Experiments are under way
to further investigate the AGO1 IRES biological function.
Page 173
EMBO Conference Series: Protein Synthesis and Translational Control
138
Translational homeostasis through UPR-mediated massive expression of
4E-BP1 in serous exocrine tissues
Charline Lasfargues, Yvan Martineau, David Müller, Steven Konieczny, Corinne Bousquet,
Stéphane Pyronnet
INSERM CRCT, France
Presenter: Charline Lasfargues
The relative amount of 4E-BP1 in mammals varies drastically among organs suggesting that
certain tissues require a stronger capacity of cap-dependent translational control than others.
Consistently, we show here that serous exocrine cell lineages specialized in the massive
production of proteins destined to secretion (including pancreatic or salivary acinar cells and
Paneth cells of the intestine) express 4E-BP1 mRNA and protein to levels far exceeding what is
found in other cell types. The robust expression of 4E-BP1 in these cells appears not
dependent on organ-specific transcription factors (such as Mist1), but instead correlates with a
permanent but physiological induction of the unfolded protein response (UPR). UPR and
4E-BP1 inductions can be recapitulated in established acinar cell lines and in ex-vivo cultures
of human acinar cells exposed to chemical UPR inducers such as tunicamycine. UPR
induction stimulates the production of the transcription factor ATF4 which in turn, activates
4E-BP1 gene transcription. These data suggest that the strong demand on protein folding
capability in the endoplasmic reticulum of serous exocrine cells generates a physiological UPR
which maintains high levels of 4E-BP1 gene transcription. In turn, 4E-BP1 provides a
physiological mean of translational control to cells whose function is to rapidly and massively
synthesize and secrete proteins on demand.
Page 174
Poster Abstracts
139
Sucrose dependent stalling of ribosomes on the bZIP11 mRNA
Jeroen Lastdrager, Jolanda Schuurmans, Johannes Hanson
Utrecht University, The Netherlands
Presenter: Jeroen Lastdrager
Sugar levels reflect the energy status of plants, and function as signaling molecules affecting
growth and development. In response to high sucrose levels, the Arabidopsis thaliana bZIP11
transcription factor is translationally repressed. bZIP11 regulates genes encoding key enzymes
in primary metabolism and is thereby involved in plant development. Current knowledge on
molecular mechanisms involved in the perception of sugars and the downstream signaling
pathways is limited. The 5’-leader of the bZIP11 mRNA harbors four upstream open reading
frames (uORFs). Only in the presence of sucrose, ribosomes translating the leader sequence
are stalled upon the transcript, and translation of the main ORF is prevented. The sucrose
control (SC) peptide, encoded by uORF2, is essential for the sucrose induced repression of
translation (SIRT) of bZIP11. Our study aims to understand the repression mechanism by
identifying proteins that interact with the SC-peptide in the presence of sucrose. Transgenic
Arabidopsis plants expressing a functional N-terminal FLAG-tagged SC-peptide have been
developed, allowing the enrichment and identification of interacting proteins. A yeast 2 hybrid
screening approach identified a TPR-containing protein, involved in the regulation of protein
translation and cell division, as an interactor of the SC-peptide. The Arabidopsis thaliana Target
of Rapamycin (TOR) kinase is as well involved in the regulation of bZIP11 translation, indicated
by reduced bZIP11 translation in TOR RNAi silenced plants. Sugars affect the protein
composition of ribosomes in Arabidopsis, therefore the roles of ribosomal protein paralogs are
explored through screening of knock-out mutants. Understanding the regulation of bZIP11
translation gives insight in how plants adapt metabolism to their energy status.
Page 175
EMBO Conference Series: Protein Synthesis and Translational Control
140
Chloroplast Ribosome RNA association
Julia Legen1, Anne Giese1, Lars Scharff2, Ralph Bock2, Christian Schmitz-Linneweber1
1 Humboldt University Berlin, Germany
2 Max Planck Institute Golm, Germany
Presenter: Julia Legen
Chloroplast gene expression is characterized by a multitude of post-transcriptional processing
steps, but regulatory dominance is believed to be exerted at the translational level. To date,
ribosome-association of chloroplast mRNAs has been assessed only for selected genes, while
whole-genome approaches were lacking. Here, we developed an assay to immunologically
enrich ribosome-associated RNAs and detect them by high-throughput analysis. To this end,
tagged ribosomal subunits were expressed both from the nuclear and the chloroplast genome
in Nicotiana tabacum. Tagged ribosomal proteins were precipitated from chloroplast extracts
and co-precipitated RNA was analyzed with a whole-chloroplast genome tiling microarray. Our
data show that light signals impact association of RNA with ribosomes. Furthermore, we found
that standard biochemical purification of polysomes enrich for overlapping, but not identical
RNA pools relative to our ribosome immunoprecipitation protocol.
Page 176
Poster Abstracts
141
Interaction of EF-G with L7/12 on the ribosome monitored in real time using
FRET
Anja Lehweß-Litzmann, Carlos da Cunha, Peske Frank, Wolfgang Wintermeyer,
Marina Rodnina
MPI for Biophysical Chemistry, Germany
Presenter: Anja Lehweß-Litzmann
During translation elongation cycle, the elongation factor G (EF-G), a five-domain GTPase,
catalyzes translocation of the tRNAs and mRNA through the ribosome. The ribosomal protein
L7/12 is thought to be the first binding partner for EF-G, recruiting the factor to the ribosome.
In E. coli, four copies of L7/12 form together with the ribosomal proteins L10 the L12 stalk
complex. The N-terminal domains of L7/12 are bound to L10, whereas the C-terminal domain
of L7/12 is connected by a flexible linker. The C-terminal domain acts as a mobile element
which interacts with the factors and is supposed to guide factors like EF-G to the ribosome.
During the interaction with the ribosome conformational changes do not only occur in L7/12
but also in EF-G. L7/12 strongly stimulates the GTP hydrolysis by EF-G; however, the interplay
between GTP hydrolysis and structural rearrangements is unknown. We studied the
conformational dynamics of the interaction of EF-G with L7/12 upon binding of the factor to
pre-translocation complexes as well as to non-translating ribosomes. This was done via
stopped flow rapid kinetics approach monitoring FRET between labels on L7/12 and EF-G.
Antibiotics and non-hydrolyzable GTP-analogues were used to assign the multiple phases of
the progress curves to different events during catalysis and to determine their order. The data
confirm that L7/12 acts as a primary binder for EF-G recruitment, the complex persists until the
late stages of translocation, and the two proteins separate concomitant to the dissociation of
EF-G after translocation. Thus, the L7/12–EF-G complex appears to be much more long-lived
than the complex of L7/12 with EF-Tu, which seems to form only transiently.
Page 177
EMBO Conference Series: Protein Synthesis and Translational Control
142
Translation control of TAK1 mRNA by hnRNP K modulates LPS-induced
macrophage activation
Anke Liepelt, Jana C. Mossanen, Bernd Denecke, Felix Heymann, Frank Tacke, Gernot
Marx, Dirk H. Ostareck, Antje Ostareck-Lederer
University Hospital, RWTH Aachen, Germany
Presenter: Anke Liepelt
Macrophage activation by bacterial lipopolysaccharides (LPS) is induced through Toll-like
receptor 4 (TLR4). The synthesis and activity of TLR4 downstream signalling molecules
modulates the expression of pro- and anti-inflammatory cytokines. To address the impact of
post-transcriptional regulation on that process we performed RIP-Chip analysis. Differential
association of mRNAs with heterogeneous ribonucleoprotein K (hnRNP K), an mRNA-specific
translational regulator, was studied in non-induced and LPS-activated macrophages. A nalysis
of potential hnRNP K targets revealed an enrichment of mRNAs encoding TLR4 downstream
kinases and their modulators. We focused our studies on TGF- β activated kinase 1 (TAK1), a
central player in TLR4 signalling. HnRNP K interacts specifically with a TAK1 mRNA 3’UTR
element in RNA-binding assays. Silencing of hnRNP K does not affect TAK1 mRNA synthesis
and stability, but enhances TAK1 mRNA translation, resulting in elevated TNF- α , IL-1 β and
IL-10 mRNA expression. Our data suggest that in non-induced macrophages TAK1 mRNA
translation is inhibited by hnRNP K bound to its 3’UTR. LPS-dependent macrophage activation
abolishes translational repression and enhanced TAK1 synthesis initiates the inflammatory
response.
Page 178
Poster Abstracts
143
The translation initiation inhibitor silvestrol is highly selective for eIF4A and
induces cell death by multiple mechanisms
Lisa Lindqvist1, Jennifer M. Chambers2, Andrew Webb1, Ingela Vikström1, Kate McArthur1, G.
Paul Savage3, Mark A. Rizzacasa2, Andreas Strasser1, David CS Huang1
1 Walter & Eliza Hall Institute of Medical Research, Australia
2 The Bio21 Institute, Australia
3 CSIRO Molecular and Health Technologies, Australia
Presenter: Lisa Lindqvist
The approval of the translation elongation inhibitor Omacetaxine mepesuccinate by the U.S.
Food and Drug Administration has validated targeting the translation machinery for the
treatment of cancer. The more recently discovered translation initiation inhibitors silvestrol and
episilvestrol, which also possess anti-cancer activity, prevent eIF4F complex formation;
however, the selectivity of these potent cytotoxic natural products remains unclear. Moreover,
the mechanism by which translation inhibitors kill cells has not been systematically
characterized, although the rapid reduction in anti-apoptotic Bcl-2 family member Mcl-1
protein levels has been suggested to be the major, possibly even the sole, driver of cell death.
[1,2] Our data reveals (epi)silvestrol is highly specific for eIF4A since the helicase was the only
protein identified by mass spectrometry to be specifically pulled down by a biotinylated form of
episilvestrol. Using genetic tools we determined that, contrary to expectations, reduction of
Mcl-1 did not significantly enhance cytotoxicity of these compounds, suggesting that it does
not have a principal role and cautions that strong correlations do not always signify causality.
Indeed, we found that silvestrol and other translation inhibitors can kill cells by mechanisms
dependant and independent of mitochondrial-mediated apoptosis (i.e. Bax and Bak). Indeed,
loss of clonogenic survival proved to be independent of the Bax/Bak-mediated apoptosis
altogether. Our findings showcase silvestrol as one of the most specific translation inhibitors to
date and illustrate the complex cell death mechanisms of translation inhibition.
References:
[1] Lucas DM et al (2009) Blood; 113: 4656–4666. 2 Chen R et al (2011) Blood; 117: 156–164.
Page 179
EMBO Conference Series: Protein Synthesis and Translational Control
144
Expression, purification and crystallization of eukaryotic translation initiation
factor 3 subunit j(eIF3j/Hcr1)
Yi Liu, Bernhard Kuhle, Ralf Ficner
Göttingen University, Germany
Presenter: Yi Liu
Eukaryotic translation initiation factor 3 subunit j(eIF3j) was shown to enhance the recruitment
of eIF3 complex to the small ribosomal subunit[1]. It binds to the aminoacyl (A) site and mRNA
entry channel of 40S through its C- terminal domain as well as eIF3b, a multi-scaffold protein,
by its N-terminal residues, respectively [2-4]. Besides its role in translation initiation, eIF3j is
required in ribosome maturation involving processing of 20S to 18S rRNA and recycling of
post-termination ribosomal complexes [5,6]. For structural characterization, Hcr1, a yeast
homologue of human eIF3j, was fused with N-terminal GST tag, overexpressed in Escherichia
coli, purified to homogeneity and crystallized at 293K using the sitting-drop vapour-diffusion
method. Single crystals have been obtained.
References:
[1] Fraser CS, Lee JY, Mayeur GL, et al. The j-subunit of human translation initiation factor eIF3
is required for the stable binding of eIF3 and its subcomplexes to 40 S ribosomal subunits in
vitro [J]. J Biol Chem, 2004, 279 (10):8946-8956. [2] Fraser CS, Berry KE, Hershey JW, et al.
eIF3j is located in the decoding center of the human 40S ribosomal subunit [J]. Mol Cell, 2007,
26 (6):811-819. [3] Nielsen KH, Valasek L, Sykes C, et al. Interaction of the RNP1 motif in
PRT1 with HCR1 promotes 40S binding of eukaryotic initiation factor 3 in yeast[J]. Mol Cell
Biol, 2006, 26 (8):2984-2998. [4] ElAntak L, Tzakos AG, Locker N, et al. Structure of eIF3b
RNA recognition motif and its interaction with eIF3j: structural insights into the recruitment of
eIF3b to the 40 S ribosomal subunit[J]. J Biol Chem, 2007,282(11): 8165-8174 [5] Valasek L,
Hasek J, Nielsen KH, et al. Dual function of eIF3J/Hcr1p in processing 20 S pre-rRNA and
translation initiation [J]. J Biol Chem, 2001, 276 (46):43351-43360. [6] Pisarev AV, Hellen CU,
Pestova TV. Recycling of eukaryotic posttermination ribosomal complexes [J]. Cell, 2007, 131
(2):286-299.
Page 180
Poster Abstracts
145
Systematic analysis of transcription factors downstream of insulin signaling
Ying Liu, Ville Hietakangas
University of Helsinki, Finland
Presenter: Ying Liu
Insulin/IGF signaling (IIS) regulates tissue growth, metabolism, reproduction and longevity in
response to nutritional status of a multicellular animal. The signaling pathways contributing to
IIS are rather well known, including phosphatidylinositol-3-kinase (PI3K)/AKT and TOR
pathways (Hietakangas & Cohen, 2009). Gene expression regulation plays a central role in
insulin signaling; typically hundreds of genes are either up- or down-regulated in response to
insulin in a tissue-specific manner (Rome et al. , 2003, Wong & Sul 2010). Therefore, to
understand the essence of the mechanism, it is fundamental to know the transcriptional
network downstream of IIS. However, only a handful of transcription factors (TFs) mediating
insulin-dependent gene expression are currently known. In this study, we have constructed a
Drosophila epitope tagged TFs library that can be used to overexpress the TFs in cell culture
and to detect them by epitope specific antibodies. Since phosphorylation is known to be one
of the common modes of transcription factor regulation, we utilized the mobility shift of
phosphorylated proteins on SDS-PAGE with polyacrylamide-bound Mn2+-Phos-tag (Kinoshita
et al., 2006) to detect the total phosphorylation change in response to insulin. By this method
we screened 822 TFs and yielded 11 verified hits, all of which displayed increased
phosphorylation in response to insulin. Interestingly, 5 of the 11 hits are downstream of TOR
pathway. As TOR signaling is known to promote ribosome biogenesis, we are currently
exploring the possible involvement of our hits in this pathway.
Page 181
EMBO Conference Series: Protein Synthesis and Translational Control
146
Snapshots of the mammalian initiation of protein synthesis and the mechanism
of scanning
Ivan Lomakin1, Thomas Steitz2
1 Yale University, United States of America
2 Yale University, HHMI, United States of America
Presenter: Ivan Lomakin
The sequence of events in translation initiation, as well as the individual components, have
been described by biochemical experiments, but the details of their regulation and the
underlying molecular mechanisms have yet to be determined. During translation initiation in
eukaryotes, the small ribosomal subunit binds mRNA at the 5’-end and scans in the 5' to 3’
direction to locate the initiation codon. At this point, the large subunit joins to form the 80S
initiation complex, which is now ready for the protein synthesis. This simple, yet intricate,
process is guided by multiple initiation factors. We determined the structures of three
complexes of the small ribosomal subunit that represent distinct steps in mammalian
translation initiation. These structures reveal the locations of eIF1, eIF1A, mRNA and initiator
tRNA bound to the small ribosomal subunit and provide insights into the details of translation
initiation that are specific for eukaryotes. Conformational changes associated with the
functional states that have been captured reveal the dynamics of the interactions in the P site
of the ribosome. These results have functional implications for the mechanism of mRNA
scanning.
Page 182
Poster Abstracts
147
The 5’untranslated region of the human T-cell lymphotropic virus type 1 mRNA
exhibits cap-independent translation initiation
Eduardo Olivares1, Dori M. Landry2, Carlos Caceres1, Karla Pino1, Federico Rossi1, Camilo
Navarrete3, Juan Pablo Huidobro-Toro3, Sunnie R. Thompson2, Marcelo Lopez-Lastra1
1 Escuela de Medicina, Pontificia Universidad Católica de Chile, Chile
2 University of Alabama at Brimingham, United States of America
3 Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Chile
Presenter: Eduardo Olivares
In this study we demonstrate that the 5’untranlated region (5’UTR) of the capped HTLV-1
full-length mRNA harbors an IRES. Cap-independent translational activity was demonstrated
using dual luciferase bicistronic mRNAs in rabbit reticulocyte lysate, in mammalian cell culture,
and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in the
bicistronic constructs resulted from an alternatively spliced transcript or from cryptic promoter
activity associated with the 5’UTR of the HTLV-1 mRNA was evaluated. Furthermore, we show
that the HTLV-1 IRES is dependent on ribosomal protein S25 for full activity.The HTLV-1 IRES
is sensitive to edeine. Together these findings suggest that recruitment of the initiation complex
can occur internally yet recognition of the initiation codon requires scanning ternary complexes.
Page 183
EMBO Conference Series: Protein Synthesis and Translational Control
148
The RNA chaperone protein Mex3A stimulates N-dependent translation
initiation of the ANDV SmRNA
Estefania Castillo1, Francisco Barriga de Vicente2, Loretto Solis1, Patricio Astudillo1, Eduard
Batlle2, Marcelo Lopez-Lastra1
1 Escuela de Medicina, Pontificia Universidad Católica de Chile, Chile
2 Colorectal Cancer Lab, Institute for Research in Biomedicine (IRB) Barcelona, Spain
Presenter: Marcelo Lopez-Lastra
Eukaryotic messenger RNAs (mRNAs) present a 5’cap structure, and most a 3’poly(A) tail.
Both elements participate in the process of translation initiation by mediating mRNA
circularization. The translation initiation factor 4G (eIF4G) allows mRNA circularization by
binding to the cap-binding protein eIF4E, and the poly(A) binding protein, PABP. In contrast to
most cellular mRNAs, the capped Small segment mRNA (SmRNA) of the Andes Hantavirus
(ANDV) lacks a poly(A) tail. Consistent with this feature, translation initiation of the ANDV
SmRNA is PABP independent. Additionally, the hantaviral N protein has been shown to replace
eIF4F. We hypothesized that during translation initiation, the circularization of ANDV SmRNA is
mediated by the cap-binding viral protein N and cellular proteins that interact with the viral
3’UTR. Through an RNA affinity chromatography approach, ANDV 3’UTR-protein complexes
were isolated. The proteins associated with the ANDV 3’UTR were identified by mass
spectrometry. The RNA chaperone Mex3A was identified as part of the ANDV 3’UTR-protein
complex. Next, the possible role of Mex3A in the translation of ANDV SmRNA was evaluated in
the absence and in the presence of the ANDV N protein. Results show that during translation
initiation, Mex3A binds the 3’UTR sequence enabling N-dependent translation initiation from
the viral mRNA. Results provide additional evidence in support for a closed-loop model of
translation initiation and describe a novel interaction established in a viral mRNA that substitute
for the molecular bridging contacts that occur in normal cellular mRNAs.
Page 184
Poster Abstracts
149
Homeostatic regulation of ribosomal protein synthesis involves changes in
translation elongation
Fabrizio Loreni1, Angelo Gismondi1, Giada Juli1, Vinay Sagar1, Gaia Lisi1, Lidia Chellini1,
Valentina Iadevaia2, Christopher Proud2, Sara Caldarola1
1 University of Rome Tor Vergata, Italy
2 University of Southampton, United Kingdom
Presenter: Fabrizio Loreni
The translation of TOP mRNAs is regulated by growth signals through the mTORC1 pathway,
although the biochemical details underlying this control are not clearly defined. In particular, the
fraction of TOP mRNAs associated with polysomes is always lower than the average of other
messengers. In this report we propose that the low translational efficiency of TOP mRNAs is a
necessary feature for a homeostatic control of ribosome synthesis that involves regulation of
translation elongation. In fact we observe that alteration of ribosome production induces an
inhibition of translation elongation that leads to enhanced association of TOP mRNAs on
polysomes. This condition allows cellular resources to be directed towards the production of
ribosomal components to restore a sufficient level of ribosomes. The inhibition of elongation
observed in response to alteration of ribosome synthesis (ribosomal stress) correlates with an
increase of eEF2 phosphorylation and activation of eEF2 kinase.
Page 185
EMBO Conference Series: Protein Synthesis and Translational Control
150
Identification and comparative analysis of in vivo phosphorylation of eEF1A1
and eEF1A2
Garry Corthals1, Tetyana Lukash2, Boris Negrutskii2
1 Translational Proteomics Group, Turku Centre for Biotechnology, Finland
2 Institute of molecular biology and genetics, Ukraine
Presenter: Garry Corthals
Higher eukaryotic translation elongation factor 1A (eEF1A)*GTP catalyses codon-dependent
binding of aminoacyl-tRNA to the A site of 80S ribosome. The two eEF1A isoforms (eEF1A1
and eEF1A2) share 92% sequence identity. eEF1A1 is expressed in all tissues throughout
development, eEF1A2 - in skeletal muscle, brain and heart. These tissues initially express
eEF1A1, but switching towards expression of the eEF1A2 occurs in the early postnatal life.
Importantly, the appearance of eEF1A2 in tissues in which this variant is not normally
expressed can be coupled to cancer development. We reasoned that the background for the
functional difference of eEF1A1 and eEF1A2 might lie in differences in posttranslational
modifications, especially phosphorylation. To check this hypothesis, we analyzed
phosphopeptides of eEF1A isolated from different cell lines and isoform-specific tissues - liver
and muscle by using TiO2 enrichment technique together with Q Exactive LC-MS/MS system.
Mass spectrometry analysis identified eight phosphorylation sites on eEF1A in vivo and
confirmed previously shown sites. Two of these sites (Y177 and S316) are in eEF1A1-specific
positions, three (S175, T239, S393) – in eEF1A2-specific positions. Four of these sites (S316,
S175, T239, S393) were identified first time in our study. All obtained spectra will be used
further for selected reaction monitoring (SRM) analysis. This high sensitive technique enables
targeted, quantitative and systematic monitoring of proteins and PTMs via their constituent
peptides. To establish SRM assays for the protein, first we selected peptides that produced
optimal MS signal responses, as these cannot be predetermined based on sequence. The
quantitative changes that occur over time for PTMs can potentially be matched to the
quantitative fluctuation of binding partners, thereby help us to resolve the mechanism
underlying the putative functional differences between the isoforms.
Page 186
Poster Abstracts
151
Decapping activity of human Nudt16 enzyme towards short
ribooligonucleotides capped with novel monomethylated (MMG) and
trimethylated (TMG) cap analogs modified in the triphosphate chain with S or
NH
Maciej Lukaszewicz, Natalia Stelmaszuk, Elzbieta Bojarska, Malgorzata Zytek, Jacek
Jemielity, Joanna Kowalska, Edward Darzynkiewicz
University of Warsaw, Poland
Presenter: Maciej Lukaszewicz
Decapping is a critical step in multiple pathways of mRNA degradation. Nudt16 is one of the
recently identified mRNA decapping enzyme that, similarly to Dcp2, belongs to the NUDIX
protein family and cuts m7GDP from mRNAs. Nudt16 also hydrolyses TMG-capped RNAs
(e.g. U8 snoRNA) similarly to its X.leavis homolog X29, and thus could play role in cellular
stability of both, MMG and TMG-capped RNAs. Previously we synthesized mRNAs with MMG
caps modified within the triphosphate bridge (with S, NH, Se and BH3) that were resistant to
decapping by hDcp2. We now describe TMG dinucleotide cap analogs substituted at the
β-phosphate position of the triphosphate bridge with S (TMGppSpG -D1 and –D2) or at either
the α-β or β-γ position with NH (TMGppNHpG and TMGpNHppG, respectively). All tested
TMG-cap analogs were co-transcriptionally incorporated by SP6 RNA polymerase into short
ribooligonucleotide transcripts. Preliminary data show that TMG-capped transcripts as short as
5 nucleotides in length are specifically decapped in vitro with hNudt16 enzyme. Transcripts
capped with TMGppSpG (D1), TMGppSpG (D2) and TMGppNHpG cap analos were resistant
to decapping with hNUDT16, in contrast to transcripts capped with TMGpNHppG that were
effectively hydrolysed.
Page 187
EMBO Conference Series: Protein Synthesis and Translational Control
152
cis regulatory elements of oskar mRNA regulate translation in trans
Paul Macdonald, Goheun Kim, Matt Kanke, Young Hee Ryu
University of Texas at Austin, United States of America
Presenter: Paul Macdonald
Translation of mRNAs is often regulated by cis acting control elements, commonly positioned
in the 5’ or 3’ untranslated regions (UTRs). The Drosophila oskar mRNA encodes a protein
required for embryonic body patterning and germ cell formation. Control elements that mediate
repression of the oskar mRNA are found in the 3‘UTR, while activating elements appear in both
5’ and 3’ UTRs. The key repressive control elements are the Bruno Response Elements, or
BREs, which are bound by the Bruno protein. BREs appear in two separate regions in the 3’
UTR, one close to the oskar coding region and the other close to the mRNA 3’ end. The 3’
proximal BREs, as well as other types of Bruno binding sites in the same region, play an
additional role in activation of translation. Defects associated with mutation of BREs and other
Bruno binding sites are assessed in transgenic flies, in which only the mutant form of the oskar
mRNA is present. These defects include loss of posterior patterning when activation of oskar
mRNA translation is disrupted, and excess posterior patterning when repression is disrupted.
Remarkably, both types of defect are largely or completely rescued when an oskar mRNA with
wild type regulatory elements but unable to make a functional Oskar protein is also present.
The two most plausible explanations of this phenomenon invoke (i) competition for regulatory
factors or (ii) cis regulatory elements also acting in trans . The behavior of mRNAs that would
be expected to be effective competitors for regulatory factors, yet fail to rescue the regulatory
defects, argues strongly against the first model. For the regulation in trans model, intimate
association of different molecules of oskar mRNA could facilitate action of regulators bound to
one mRNA on another mRNA. Interfering with the ability of oskar mRNAs to associate with one
another disrupts rescue, supporting the model of translational regulation in trans.
Page 188
Poster Abstracts
153
The role of the unfolded protein response in neurodegeneration: a new target
for therapy of these disorders.
Giovanna Mallucci
MRC Toxicology Unit, United Kingdom
Presenter: Giovanna Mallucci
During prion disease, rising levels of misfolded prion protein (PrP) generated by prion
replication lead to sustained over-activation of the branch of the unfolded protein response
(UPR) controlling the initiation of protein synthesis. This causes persistent repression of
translation, resulting in the catastrophic loss of critical proteins and hence synaptic failure and
neuronal death. Localised genetic manipulation of this pathway focally rescues translational
shutdown and prevents neurodegeneration in prion-diseased mice, leading us to predict that
its pharmacological inhibition would give widespread neuroprotection by the same mechanism.
We have now shown that oral treatment with a specific inhibitor of the kinase PERK, a key
mediator of this pathway, prevents development of clinical prion disease and produces marked
neuroprotection throughout the brain in mice treated both at pre-clinical stage, and later in
disease, when behavioral signs had emerged. Critically, the compound acts downstream, and
independently, of the primary pathogenic process of prion replication and is effective despite
continuing accumulation of PrP. The compound also prevents neurodegeneration in a tau
transgenic mouse model. Thus the data support drug development programs targeting PERK
and other members of this pathway for treatment of prion, and potentially other UPR-inducing,
neurodegenerative diseases.
Page 189
EMBO Conference Series: Protein Synthesis and Translational Control
154
Exploring role of translation initiation in stochastic gene expression
Naglis Malys, Vinoy Ramachandran, John McCarthy
University of Warwick, United Kingdom
Presenter: Naglis Malys
Isogenic cell populations are subject to phenotypic differences as a result of stochastic
variations in the abundance of cellular components. Stochasticity has roles in cellular
differentiation, stress responses, fate decisions and potentially evolutionary transitions. It plays
an important role in gene expression. Studies on prokaryotic gene expression have suggested
that translation initiation is an important process in generating noise in protein abundance. It is
now evident that process of translation can be controlled by a variety of extrinsic and intrinsic
determinants. We have developed tools to study stochastic cell-to-cell variation in gene
expression at both the transcriptional and translational levels using Saccharomyces cerevisiae
as a model organism. To achieve this, we have utilised synthetic biology tools (e.g. luciferase
and fluorescent protein reporters), measurement technologies (e.g. flow-cytometry,
microscopy and cell management platforms), and methods for absolute quantification of
mRNA at single-cell level. In the present work, noise determinants, such as a copy number of
gene (and mRNA transcripts) and mRNA secondary structure at the start of the gene, were
evaluated by controlling gene expression via the introduction of bottlenecks into the expression
system modulating transcription and translation initiation. The noise has been measured using
yeast an enhanced green fluorescent protein (yEGFP)-based reporter. A greater noise level
was observed in an isogenic cell population containing a single copy of the gene than that
observed in a cell population with multiple copies of the same gene. Introducing mRNA stem loop structure with altered thermodynamic stability at the start codon AUG of reporters yielded
a reduction in translation initiation efficiency of the cell population, which correlated negatively
with the noise increase in protein abundance at the individual cell level.
Page 190
Poster Abstracts
155
Reinitiation-supporting protein RISP interacts with eIF3 and eIF2
Eder Mancera-Martinez, Lyubov Ryabova
Institut de Biologie Moléculaire de Plantes, France
Presenter: Eder Mancera-Martinez
Reinitiation supporting protein RISP was discovered as a component of the cell translation
machinery that is employed for translation of the Cauliflower mosaic virus 35S polycistronic
RNA under control of translation transactivator/ viroplasmin (TAV). TAV activates the
mechanism of reinitiation after long ORF translation via interaction with translation initiation
factor 3 (eIF3) and RISP, their recruitment into polysomes to ensure reinitiation at a further
downstream ORF on the same mRNA. According to our in vitro data, the complex between
RISP and eIF3 can associate with the 40S ribosomal subunit (40S), while RISP failed to join the
eIF3-40S complex. CaMV is the first known virus able to interact directly with target of
rapamycin (TOR) protein kinase and triggers TOR signalling pathway activation towards
translation. RISP was identified, as a novel target of the TOR signalling pathway, and its
phosphorylation at Ser 267 is required to promote TAV-activated reinitiation. However, RISP
function in cellular translation as well as in TAV-mediated transactivation is still under
investigation. Here we report that in addition to eIF3 binding, RISP can associate with wheat
eIF2 in vitro and be specifically immunoprecipitated by anti eIF2α antibodies in planta. The
interaction was further confirmed by the yeast two-hybrid system, which revealed the eIF2
subunit β as a RISP specific target. According to our preliminary data, phosphorylation of RISP
is not critical for complex formation between RISP and eIF2β. However, the RISP
phosphorylation knockout (RISP-S267A) binds eIF2β more efficient than its phosphorylation
mimic (RISP-S267D). Our current model is that RISP may bridge eIF3 and eIF2 within the 43S
pre-initiation complex to promote ternary complex (eIF2/GTP/Met-tRNAi Met) recruitment to
40S.
Page 191
EMBO Conference Series: Protein Synthesis and Translational Control
156
Internal ribosome entry site-mediated translational regulation of mammalian
target of rapamycin (mTOR) in stress conditions
Ana Marques-Ramos, Rafaela Lacerda, Alexandre Teixeira, Luísa Romão
Instituto Nacional de Saúde Dr. Ricardo Jorge, Portugal
Presenter: Ana Marques-Ramos
Regulation of translation is a key mechanism by which cells and organisms can rapidly change
their gene expression patterns in response to extra- and intracellular stimuli. Translational
control can occur on a global basis by modifications of the basic translation machinery, or
selectively target defined subsets of mRNAs to maintain synthesis of certain proteins required
either for stress responses or to aid recovery from the stress condition. These pathways are
evolutionary conserved and have been shown to significantly impact translation in organisms
as diverse as yeast and humans. In many cases, features in the 5’ untranslated regions
(5’UTRs) of the corresponding mRNAs, such as internal ribosome entry sites (IRESs) are
important for them to evade global repression of translation. IRES-mediated translation is an
alternative to the cap-dependent mechanism of translation initiation that involves direct
recruitment of the ribosome to the vicinity of the initiation codon and may require several IRES
trans-acting proteins. Mammalian target of rapamycin (mTOR) is a highly conserved kinase that
is responsive to several cellular stimuli. Deregulation of mTOR signalling is implicated in major
diseases, mainly due to its role in regulating protein synthesis. The main mTOR targets are
proteins responsible for ribosome recruitment to the mRNA, thus, a specific inhibitor of mTOR,
for example rapamycin, leads to global inhibition of translation. Knowing that in stress
conditions such as hypoxia, overall protein synthesis is reduced, but synthesis of mTOR
protein is not inhibited, we hypothesized that mTOR 5’UTR harbours an IRES allowing
cap-independent synthesis of mTOR protein in stress conditions. By using dicistronic reporter
plasmids we have tested and confirmed this hypothesis. Our findings provide a framework for
understanding how translational reprogramming of mTOR mRNA is regulated in response to
cellular stress conditions.
Page 192
Poster Abstracts
157
Involvement of the RNA-binding protein Gemin5 in IRES-dependent translation
David Piñeiro, Javier Fernandez, Jorge Ramajo, Rosario Francisco, Encarna
Martinez-Salas
Centro de Biologia Molecular Severo Ochoa, Spain
Presenter: Encarna Martinez-Salas
Internal ribosome entry site (IRES) elements govern protein synthesis in various RNA viruses,
including picornaviruses. This process requires the interaction of the IRES element with several
eukaryotic initiation factors and RNA-binding proteins termed IRES transacting factors (ITAFs).
A riboproteomic approach carried out with two unrelated IRES elements, hepatitis C (HCV) and
foot-and-mouth disease virus (FMDV), revealed the interaction of novel IRES-interacting
factors, among them Gemin5, a predominantly cytoplasmic protein involved in the assembly of
the SMN complex and thus, in the biogenesis of the small nuclear ribonucleoproteins. Gemin5
acts as a negative regulator of IRES-dependent translation, revealing a new role for this protein
in translation control. Immunoprecipitation of UV-crosslinked complexes showed that the
binding site of Gemin5 within the FMDV IRES resides in the hairpin of domain 5. Mapping the
regions of Gemin5 involved in IRES-interaction by UV-crosslink and RNA-binding assays
indicated that the most C-terminal region of the protein is sufficient to bind directly with domain
5 of the IRES. Furthermore, this region down-regulates translation of a bicistronic RNA both in
cell free systems as well as in Gemin5-depleted cells. These data show that the C-terminal end
of Gemin5 plays a critical role in controlling the interaction of RNA-binding proteins with the
FMDV IRES, as well as in its function as a negative regulator of translation.
Page 193
EMBO Conference Series: Protein Synthesis and Translational Control
158
Structure of the archaeal translation initiation complex
Pierre-Damien Coureux1, Emmanuelle Schmitt1, Eric Larquet2, Lionel Cladiere1,
Jean-François Menetret3, Bruno Klaholz4, Yves Mechulam1
1 Ecole Polytechnique-CNRS, Palaiseau, France
2 LEBS, CNRS, Gif sur Yvette, France
3 IGBMC, CNRS, Inserm, Université de Strasbourg, France
4 Institute of Genetics and of Molecular and Cellular Biology, France
Presenter: Yves Mechulam
Eukaryotic and archaeal translation initiation complexes have in common a functional core
containing the mRNA, the ternary initiation complex (e/aIF2:GTP:Met-tRNAiMet), e/aIF1 and
e/aIF1A bound to the small ribosomal subunit. In eukaryotes, the functional core is made more
complex by many additional factors, most of them being involved in a long-range scanning of
mRNA, necessary to decipher the initiation codon. In archaea, long-range scanning does not
occur thanks to the occurrence of Shine-Dalgarno sequences or of very short 5’UTR on
mRNA. Concomitantly, archaeal translation initiation only requires the core complexes. Two
archaeal translation initiation complexes, representing distinct steps of translation initiation
were studied using cryo-electron microscopy. The two structures reveal the locations of
aIF2:Met-tRNAiMet, aIF1 and aIF1A bound to the archaeal 30S subunit and provide insights
into translation initiation steps that are common to archaea and eukaryotes.
Page 194
Poster Abstracts
159
Influence of translation factor activities on start site selection
Diane Barth-Baus, William Merrick
Case Western Reserve University, United States of America
Presenter: William Merrick
Current literature using biochemical assays, structural analyses and genetic manipulations has
reported that the key factors associated with the faithful matching of the initiator met-tRNA to
the start codon AUG are eIF1, eIF1A and eIF5. However, these findings were in each case
based upon the utilization of a single mRNA, perhaps with variations. In an effort to evaluate
this general finding, we tested six different mRNAs. Our results confirm that these three
proteins are important for start site selection. However, two additional findings would not have
been predicted. The first is that eIF1 plays a major role in selecting against start codons that
are in close proximity to the 5’ end of the mRNA (i.e. less than 21 nucleotides). Secondly, the
addition of eIF5B had nearly the same affect as the addition of eIF5. This is unexpected given
the different roles that eIF5 and eIF5B have been proposed to play in the 80S initiation
pathway. Finally, although many of the mRNAs appear to respond qualitatively in a similar
manner, the quantitative differences noted suggest that there is still some mRNA specific
character to our findings. These results are discussed in a model that envisages reversible
steps in protein synthesis initiation.
Page 195
EMBO Conference Series: Protein Synthesis and Translational Control
160
GWIPS-viz: Development of a dedicated ribo-seq genome browser
Audrey Michel1, Gearoid Fox2, Christof De Bo3, Patrick BF O'Connor1, Pavel Baranov1
1 University College Cork, Ireland
2 University College Cork, University College Dublin, Ireland
3 University College Cork, HOWEST University College West Flanders, Belgium
Presenter: Audrey Michel
Ribosome profiling (ribo-seq) is a recently developed technique that provides Genome Wide
Information on Protein Synthesis (GWIPS) in vivo. The deep sequencing of ribosome protected
mRNA fragments yields a snapshot of the ribosome distribution on all mRNA transcripts
present in the cell. Since its inception, ribo-seq has been carried out in a number of eukaryotic
and prokaryotic organisms. As the use of ribo-seq in experimental studies is growing at an
accelerated pace, there is a pertinent demand for a dedicated ribo-seq genome browser.
Therefore we have developed GWIPS-viz which is an online genome browser for viewing
published ribosome profiling data (http://gwips.ucc.ie/). GWIPS-viz is based on the UCSC
Genome Browser. Ribo-seq tracks coupled with mRNA-seq tracks are currently available for
H. sapiens, M. musculus, D. rerio, C. elegans, S. cerevisiae, E. coli and B. subtilis. Although
still in early stage development, our objective is to continue incorporating ribo-seq datasets so
that the wider community can easily and readily view published ribosome profiling information
without the need to process and align the ribo-seq data themselves.
Page 196
Poster Abstracts
161
Human Dbp5 regulates translation termination activity of eRF1
Tatiana Mikhaylova, Elena Alkalaeva
Engelhardt Institute of Molecular Biology RAS, Russian Federation
Presenter: Tatiana Mikhaylova
The DEAD-box RNA helicase Dbp5 is an essential mRNA export factor. Additionally, a novel
important function of Dbp5 was identified in translation termination in yeast. But mechanism of
Dbp5 activity during termination of translation remains unclear. We have cloned human
homolog of Dbp5 (Ddx19) and produced recombinant protein in E. coli. Helicase activity of
recombinant human Dbp5 was demonstrated using luciferase based assay. We have shown
that only 20% of human Dbp5 binds eRF1 in the presence and absence of ATP in vitro. We
suppose that for effective Dbp5-eRF1 complex formation additional factors are required.
Activity of eRF1-Dbp5 complex in translation termination was tested in reconstituted in vitro
eukaryotic translation system. We have shown that Dbp5 significantly decreases activity of
eRF1 in the absence of eRF3. Probably binding of eRF1 with Dbp5 during translation changes
conformation of the protein and regulates peptide release.
Page 197
EMBO Conference Series: Protein Synthesis and Translational Control
162
Translationally silent but tuneable G-quadruplex structure at the VEGF IRES-A
Stefania Millevoi, Benjamin Morel, Alexandre Dubrac, Anne Cammas, Christian Touriol,
Hervé Prats
INSERM, France
Presenter: Stefania Millevoi
RNA G-quadruplexes have been recently identified as critical regulator of gene expression. At
the translational level, RNA G-quadruplexes act as general inhibitors of the initiation step of
cap-dependent mRNA translation. However, few cases have been reported where
G-quadruplexes are associated to RNA elements that mediate internal ribosome entry sites
(IRES) activity. Functional analysis of a specific set of mutations in the G-quadruplex forming
motif within the human VEGF IRES-A has recently revealed that this structure is essential for
IRES-mediated translation. Here we have analyzed the structure/function relationship of the
VEGF G-quadruplex motif by using different nucleotidic variants of this specific combination of
mutations. We demonstrate that the G-quadruplex within the VEGF IRES-A is dispensable for
IRES function and activation in stress conditions. However, stabilization of the VEGF
G-quadruplex results in inhibition of IRES-A-mediated translation. Our results uncover the
existence of functionally silent G-quadruplex structures that are susceptible to be converted
into efficient modulators of cap-independent mRNA translation.
Page 198
Poster Abstracts
163
A dependancy on eIF2B Phosphorylation for cell cycle progression in stressed
yeast
Sarah Mohammad-Qureshi1, Rehana Kousar1, Rebecca Sanderson2, Graham Pavitt1
1 University of Manchester, United Kingdom
2 Integrated Genetics, Labcorp speciality testing group, United States of America
Presenter: Sarah Mohammad-Qureshi
Reactivation of eIF2 following each translation initiation event is dependent on eIF2B function
and presents a key point of translational regulation. Indirect inhibition of eIF2B through the
phosphorylation of eIF2α during cellular stress is well established. This is a highly conserved
process in all eukaryotic cells, in S. cerevisiae the eIF2 kinase Gcn2p is activated by amino
acid starvation. Mammalian eIF2B is also regulated through direct phosphorylation. Here we
demonstrate how phosphorylation at Ser435 of the largest subunit of yeast eIF2B (encoded by
GCD6) can impact on cellular growth and stress responses. A S435A mutation causes growth
arrest on plate assays in response to temperature and nutrient stresses, conversely the S435E
mutant is only sensitive to the inhibitors of the TOR pathway. Data suggests the S435A
mutation is overriding the general control response of amino acid starvation. Microscopy
reveals the S435A mutant yeast arrest early in the cell cycle under different stresses, as does
S435E in response to rapamycin. By following α-factor synchronised cells during their release
into fresh medium, we find Ser435 is necessary for timely progression of cell cycle stages,
seen by FACS analysis. rapamycin-treated S435A yeast demonstrate failure to regulate cell
size, consistant with delayed START. Using phospho-specific antibodies, we observe
phosphorylation of Ser435 at α-factor arrest that declines and peaks again at 90mins
post-release, correlating with the onset of another cell cycle. In vitro nucleotide exchange
assays are currently being carried out to determine the impact of Ser435 mutations on eIF2B
activity. We propose that timely cell cycle progression is dependent on the phosphorylation of
eIF2B at the onset of a new cell cycle, representing a critical signal for cells to re-enter the cell
cycle after a stress-induced G1 pause.
Page 199
EMBO Conference Series: Protein Synthesis and Translational Control
164
Selective mRNA translation controls erythropoiesis: regulation and function of
the RNA-binding protein Csde1
Kat Moore1, Rastislav Horos2, Marieke von Lindern3
1 Sanquin Research, The Netherlands
2 EMBL Heidelberg, Germany
3 Sanquin Research and Landsteiner Lab. AMC/UvA, The Netherlands
Presenter: Kat Moore
Erythropoiesis is heavily regulated at the level of mRNA translation, allowing a rapid response
to environmental factors. Diamond-Blackfan Anemia (DBA), a dominantly inherited anemia, is
caused by haploinsufficiency of one of several ribosomal proteins. DBA was modeled by
reducing expression of Rps19 or Rpl11, and genome wide mRNA recruitment to
polyribosomes was analyzed. This revealed several IRES-mediated transcripts that were
specifically lost from polyribosomes in the DBA model when compared to control conditions.
Among the affected transcripts was Csde1 (Cold-shock domain protein E1), an RNA-binding
protein known to bind the IRES of its own transcript. Csde1 is 200-fold upregulated in
erythroblasts relative to other hematopoietic cells. Knockdown of Csde1 impaired proliferation
and differentiation of erythroblasts. These findings prompted us to investigate the role of Csde1
We employed mouse erythroleukemic (MEL) cells
in the regulation of erythropoiesis.
expressing the prokaryotic biotin ligase BirA and in vivo biotinylated Csde1 at levels that are
comparable with endogenous Csde1. Biotagged Csde1 was enriched on streptavidine beads,
the associated mRNA was isolated and analysed by RT-PCR and on expression arrays.
Whereas Csde1 specifically bound its known targets, Csde1 and Apaf1 mRNA, it did not bind
the IRES-containing mRNA Bag1 and was reduced in polyribosomes of DBA models. Thus,
decreased expression of Csde1 is not responsible for reduced IRES-dependent translation in
DBA. Genome-wide analysis of transcripts bound by Csde1 uncovered several mRNAs with
Csde1 recognition sites in their 5’ or 3’ UTR. Currently, we are extending this profiling of
Csde1-bound mRNAs and investigating protein binding partners of Csde1. From there,
knockdown studies can be employed to determine which transcripts and proteins bound by
Csde1 mediate the essential function of Csde1 in erythropoiesis.
Page 200
Poster Abstracts
165
The role of cap-independent mRNA translation in germ cell fate decisions
J. Kaitlin Morrison, Andrew Friday, Vince Contreras, Brett Keiper
Brody School of Medicine at East Carolina University, United States of America
Presenter: J. Kaitlin Morrison
During translation initiation eIF4GI provides a scaffolding complex allowing for the association
of initiation factors and ribosomes on recruited mRNAs. We have previously shown that C.
elegans caspase, CED-3, cleaves eIF4GI (IFG-1 p170) during apoptosis and removes the
eIF4E and PABP binding domains, preventing their involvement in translation initiation. Unlike
mammalian cells, only two eIF4G forms are found in C. elegans, IFG-1 p170 (cap-associated)
ad p130 (not cap-associated). Despite the decrease in cap-dependent translation, initiation on
a select subset of mRNAs becomes more efficient. Cleaved eIF4GI and its cap-independent
paralog, p97, are known to bind directly to mRNA, often via internal ribosome entry sites
(IRESs) to recruit these mRNAs for translation. Thus, during apoptosis there is a switch in the
mode of translation initiation to IRES-mediated recruitment that results in selective translation
of a specific subset of mRNAs. In the C. elegans gonad, nearly half of all cells entering meiosis
are fated for death before reaching maturity. All germ cells entering gametogenesis experience
a condensation of chromosomes, suppressing transcription. Therefore, changes in protein
expression leading to various germ cell fates result from translational control of stored maternal
mRNAs. In this study we address the translational regulation of the stress and apoptotic
related mRNAs in C. elegans: BiP, Hif-1, p53, Bcl-2 and Apaf-1. Altered translational efficiency
of these messages was observed upon genetic depletion of cap-dependent IFG-1 or
independent induction of the apoptotic pathway. Our findings suggest a physiological link
between the cap-independent mechanism and the enhanced translation of BiP and Bcl-2.
Translational activation may be integral to the stress response during the induction of germ cell
apoptosis. We are currently following up these experiments by investigating cap-independent
translation in the germ line of live worms.
Page 201
EMBO Conference Series: Protein Synthesis and Translational Control
166
4E-BP restrains eIF4E phosphorylation
David Müller1, Sally El-Khawand1, Charline Lasfargues1, Amadine Alard2, Robert J
Schneider2, Corinne Bousquet1, Stéphane Pyronnet1, Yvan Martineau1
1 INSERM, France
2 NYU School of Medicine, United States of America
Presenter: David Müller
In eukaryotes, mRNA translation is dependent on the cap-binding protein eIF4E. Through its
simultaneous interaction with the mRNA cap structure and with the ribosome-associated
eIF4G adaptor protein, eIF4E physically posits the ribosome at the 5’ extremity of capped
mRNA. eIF4E activity is regulated by phosphorylation on a unique site by the eIF4G-associated
kinase MNK. eIF4E assembly with the eIF4G-MNK sub-complex can be however antagonized
by the hypophosphorylated forms of eIF4E-binding protein (4E-BP). We show here that eIF4E
phosphorylation is dramatically affected by disruption of eIF4E-eIF4G interaction,
independently of changes in MNK expression. eIF4E phosphorylation is actually strongly
down-regulated upon eIF4G shutdown or upon sequestration by hypophosphorylated 4E-BP,
consequent to mTOR inhibition. Down-regulation of 4E-BP renders eIF4E phosphorylation
insensitive to mTOR inhibition. These data highlight the important role of 4E-BP in regulating
eIF4E phosphorylation independently of changes in MNK expression.
Page 202
Poster Abstracts
167
The role of initiator tRNA structure in transmitting the start codon recognition
signal
Antonio Munoz1, Jinsheng Dong2, Alan Hinnebusch3, Jon Lorsch1
1 Johns Hopkins University School of Medicine, United States of America
2 National Institute of Child Health and Human Development, United States of America
3 National Institues of Health, United States of America
Presenter: Antonio Munoz
The body of the initiator tRNA (tRNAi) plays a role in transmitting the start codon recognition
signal within the ribosomal pre-initiation complex (PIC) during translation initiation. In vivo,
altering elements in various domains of tRNAi results in either decreased (Sui- phenotype) or
increased (Ssu- phenotype) fidelity of start codon recognition. We have characterized the
behaviors of mutant initiator tRNAs as they may affect the transition between the
scanning-competent Pout/Open state of the PIC and the scanning-arrested Pin/Closed state.
Ssu- mutations appear to shift the equilibrium towards the Pout state, consistent with their
increased fidelity, whereas Sui- mutations seem to allow the PIC to more easily enter the
post-start codon-recognition Pin/Closed state. Single mutations in the acceptor stem produce
both Sui- phenotypes and defects in forming the eIF2-GTP-Met-tRNAi ternary complex (TC).
Restoring the base pair at the acceptor stem rescues the eIF2-GTP binding defect and
diminishes the Sui- phenotype. Anticodon stem mutants conferring the Ssu- phenotype exhibit
defects in the affinity of TC for the 40S subunit. In kinetic studies, Sui- mutations reduce off
rates, consistent with their stabilizing the closed state of the PIC, thus decreasing start codon
fidelity. The Sui- acceptor stem mutation G70A also dramatically reduces the TC binding rate,
but this is completely rescued by an Ssu- mutation in the N-terminal tail of eIF1A. Other Suimutations in the T loop and ASL display slightly slowed rates of association, indicating a
distinct mechanism in blocking rearrangement to the Pin/Closed state. Overall, our data
indicate the body of the initiator tRNA plays a key role in transmitting the signal within the PIC
of start codon recognition. This signal transmission likely involves finely-tuned movements of
and/or conformational changes in the tRNA that are modulated by dynamic interactions with
the mRNA, initiation factors, and the 40S ribosomal subunit.
Page 203
EMBO Conference Series: Protein Synthesis and Translational Control
168
Anticodon-specific replacement of tRNA isoacceptors by their orthogonal
counterparts for site-selective protein labeling in vitro
Sergey Mureev, Zakir Tnimov, Zhenling Cui, Kirill Alexandrov
University of Queensland, Australia
Presenter: Sergey Mureev
Protein biosynthetic machinery displays enormous flexibility and precision in synthesizing
polymers from very chemically diverse building blocks. Site-specific co-translational
incorporation of the FRET-forming pairs of fluorescent dyes into protein gives invaluable tools
for high-throughput studies on structure, dynamics and kinetics of individual proteins and
analysis of protein complexes by single molecule FRET Microscopy. Many attempts were
made to harness the power of the ribosome to site selectively label the proteins. Most of these
studies were focused on the expansion of the genetic code by either exploiting non-sense
codons or by using quadruplet codon combinations. However the redundancy of the genetic
code provides a large source of potentially orthogonal codons. Selective depletion of certain
tRNA isoacceptors for amino acids encoded either by mixed codon families or by the codon
families with high wobble restrictions would potentially free codons for the decoding with the
orthogonal tRNA-counterparts charged with unnatural amino acid. To this end small anti-sense
RNA hairpins targeted against the anti-codon loop were used to selectively deplete target
tRNAs from either cell-free lysate or from the native total tRNA mixture. Orthogonal tRNAs
were charged with unnatural amino acid moieties by either flexizyme (small catalytic RNA) or
using orthogonal aminoacyl tRNA synthetase with loose specificity towards the
tRNA-anticodon. We demonstrate that we can successfully pull out certain tRNA isoacceptors
causing translational pausing at specific codon position that can be reverted by addition of
t7-transcribed orthogonal tRNA counterparts.
Page 204
Poster Abstracts
169
Nutrition dependent regulation of mRNA translation during Drosophila growth
and development
Sabarish Nagarajan, Savraj Grewal
University of Calgary, Canada
Presenter: Sabarish Nagarajan
The larval period of the Drosophila life cycle is characterized by immense nutrition-dependent
growth. When nutrients, particularly amino acids (AA) are abundant, larvae increase in mass
over 200 fold in 4 days. However, upon amino acid starvation, larvae arrest their growth and
switch their metabolism to promote survival. Extensive work has described the signaling
pathways and transcriptional changes that mediate the AA effects on larval growth. In contrast,
the role of mRNA translational control on gene expression in larval growth, survival and
metabolism is less clear. Our work addresses this issue. Using polysome analysis, we saw that
upon complete AA starvation, whole larvae translation decreased within 30mins. Maximal
effects were seen within 6-18h post starvation. We next used q-PCR analysis to examine
polysome associated mRNAs in fed vs starved animals.We found that all mRNAs tested
showed reduced translation upon AA starvation, including those mRNAs containing putative
IRESs, minimal 5'UTR predicted structure, and those mRNAs whose overall transcript levels
were strongly upregulated by AA starvation. These data suggest that starvation induces a
broad, global suppression of starvation. We are performing microarray analyses of polysomal
RNA to examine this further. Extensive work has identified the Insulin/PI3K and TOR kinase
pathways as the main signaling networks that link dietary AAs to metabolism and growth in
larvae. Upon AA starvation both pathways are suppressed and growth is halted. However, we
found that genetic activation of PI3K and TOR signaling on AA-deprived animals was not
sufficient to reverse the starvation-mediated translation block. In addition, we found that
inhibition of 4EBP and activation of S6k, the two widely proposed effectors of TOR, were
insufficient to reverse the starvation-mediated suppression of translation. We are currently
investigating other pathways that link AA availability to translation.
Page 205
EMBO Conference Series: Protein Synthesis and Translational Control
170
Genome-wide translational consequences of the yeast prion [PSI+]
Agnès Baudin-Baillieu1, Rachel Legendre1, Claire Kuchly1, Isabelle Hatin1, Claire Mestdagh1,
Daniel Gautheret1, Olivier Namy2
1 Université Paris-Sud, France
2 CNRS, France
Presenter: Olivier Namy
[ PSI + ] is the prion form of the eukaryotic release factor 3 (eRF3) encoded by the SUP35 gene
in Saccharomyces cerevisiae . The conformational change impairs the termination activity of
eRF3 and consequently increasing ribosome pausing at the stop codon. This stimulates stop
codon-dependent translational errors like stop codon suppression (readthrough) and +1
frameshifting. A broad range of phenotypes is associated with the presence of [ PSI + ]. We
have previously shown that increase of PDE2 stop codon readthrough by [ PSI + ] leads to
change in cAMP intracellular concentration and a modification of the stress response. We have
also demonstrated that polyamines are under the epigenetic control of [ PSI + ]. Indeed [ PSI +
] stimulates the production of the antizyme protein by increasing +1 frameshifting efficiency at
the antizyme shifty-stop. Modification of these two metabolic pathways explains more than half
of [ PSI + ] associated phenotypes. Despite the identification of these two genes directly
regulated by [ PSI + ] t he global landscape of gene deregulation in response to translation
termination complex depletion remains to be determined. Now, we aim to identify genes
expression modified upon the appearance of [ PSI + ] to study the genetic and physiological
consequences of the presence of this prion. To reach this objective we use a ribosome
profiling approach to identify the position of all active ribosomes mapped at the resolution of a
single nucleotide within the genome. Basically this consists to extract and deep-sequence the
ribosome protected mRNA fragment. In addition to a classical transcriptomic approach this
technique provides an opportunity to study the translational status of every gene in the cell and
to observe all translational regulations occurring in a yeast cell in response to the appearance
of [ PSI + ]. This approach allowed us to identify broadest range of [ PSI + ]-induced
translational modifications than expected.
Page 206
Poster Abstracts
171
tRNA wobble uridine hypomodification disrupts protein homeostasis by
decreasing the decoding efficiency of cognate codons in vivo
Danny Nedialkova, Sebastian Leidel
MPI for Molecular Biomedicine, Germany
Presenter: Danny Nedialkova
Nucleoside modifications in tRNA are ubiquitous in all domains of life and those in the
anticodon are important for accurate codon recognition. The wobble uridine (U34) base of
eleven cytoplasmic tRNAs in eukaryotes is modified by 5-methyl derivatives (xm5). In three of
these tRNA species - tE(UUC), tK(UUU), and tQ(UUG) - U34 is also thiolated at the 2-carbon
(s2). Aberrant U34 modification is associated with increased stress sensitivity in many
organisms. In yeast, the phenotypes can be suppressed by overexpressing hypomodified
tK(UUU) and tQ(UUG), but the underlying molecular events are unknown. To characterize the in
vivo roles of U34 modifications, we examined codon-specific ribosome occupancy patterns in
wild-type yeast and strains deficient in s2 and/or xm5 addition by ribosome profiling. Strikingly,
AAA and CAA, and, to a lesser extent, GAA codons were selectively enriched in the putative A
site within ribosome footprints from the mutant strains. Increased ribosome occupancy specific
for these three codons was also evident in a C. elegans strain lacking U34 2-thiolation. Thus,
codon-specific translation defects are associated with aberrant U34 modification and this
phenomenon is conserved between yeast and nematodes. Upon analysis of gene expression
in yeast strains with hypomodified U34, we noted a significant upregulation of chaperones and
the ubiquitin-proteasome system (UPS). These data were indicative of deregulated protein
homeostasis and subsequent experiments revealed the presence of aggregated proteins in the
mutant strains. Remarkably, the degree of protein aggregation and UPS upregulation were
proportional to the extent of U34 hypomodification. Our findings indicate that in vivo, lack of
U34 modification results in translational slowdown at cognate codons and accumulation of
misfolded proteins. Perturbed protein homeostasis therefore likely underlies the phenotypes
associated with U34 modification deficiencies.
Page 207
EMBO Conference Series: Protein Synthesis and Translational Control
172
microRNA-122 dependent binding of Ago2 protein to Hepatitis C Virus RNA
associated with enhanced RNA stability and translation stimulation
Dominik Conrad1, Florian Giering1, Corinna Erfurth1, Angelina Neumann1, Carmen Fehr1,
Gunter Meister2, Michael Niepmann1
1 Justus-Liebig-University, Germany
2 University of Regensburg, Germany
Presenter: Michael Niepmann
Translation of Hepatitis C Virus (HCV) RNA is directed by an internal ribosome entry site (IRES)
in the 5´-untranslated region (5´-UTR). HCV translation is stimulated by the liver-specific
microRNA-122 (miR-122) that binds to two binding sites between the stem-loops I and II near
the 5´-end of the 5´-UTR. Here we show that Ago2 protein binds to the HCV 5´-UTR in a
miR-122-dependent manner, whereas the HCV 3´-UTR does not bind Ago2 protein. miR-122
also recruits Ago1 to the HCV 5’-UTR. Only miRNA duplex precursors of the correct length
stimulate HCV translation, indicating that the duplex miR-122 precursors are unwound by a
complex that measures their length. Insertions in the 5´-UTR that increase the distance
between the miR-122 binding sites and the IRES only slightly decrease translation stimulation
by miR-122. In contrast, partially masking the miR-122 binding sites in a stem-loop structure
impairs Ago2 binding and translation stimulation by miR-122. In an RNA decay assay, also
miR-122-mediated RNA stability contributes to HCV translation stimulation. These results
suggest that Ago2 protein is directly involved in loading miR-122 to the HCV RNA and
mediating RNA stability and translation stimulation.
Page 208
Poster Abstracts
173
High-resolution structural analysis of solvent and ion interactions with the
ribosome
Jonas Noeske, Jennifer Doudna, Jamie Cate
University of California at Berkeley, United States of America
Presenter: Jonas Noeske
The ribosome consists of a small and a large ribosomal subunit. The function of the ribosome
in protein biosynthesis requires the rotation of the two subunits with respect to each other.
This rotation is facilitated by different inter-ribosomal subunit contacts, or bridges. During
translation certain bridges break and new bridges are formed. Apart from these bridges, which
are constituted of direct rRNA and protein interactions between the two subunits, more
intersubunit interactions are thought to be mediated by water molecules. The highest
resolution ribosome structure of the intact bacterial ribosome published is at a resolution of 2.8
Angstrom. At this resolution electron density of water molecules is not sufficient to model water
molecules in the structure. We are collecting x-ray crystallographic data for a high-resolution
structure of the E. coli ribosome and have 56% complete data at 2.3 Angstrom resolution.
This resolution range enables us to assess the contribution of water molecules and metal ions
to intersubunit contacts and ribosome structure. Preliminary data suggest that key functional
regions of the ribosome seem to be free of ordered water molecules and metal ions. The
results could have fundamental impact on the general understanding of RNA-RNA interactions
in biology.
Page 209
EMBO Conference Series: Protein Synthesis and Translational Control
174
Charaterization of polyadenylation and deadenylation in the C. elegans germ
line
Marco Nousch, Christian Eckmann
MPI-CBG, Germany
Presenter: Marco Nousch
The regulation of complex gene expression programs is a fundamental process for the
development and functionality of tissues. In reproductive organs the primary mode to control
protein production occurs on the post-transcriptional level, making translational control one of
the hallmarks of germ cell development. On the molecular level, controlled changes in mRNA
3’ poly(A) tail lengths are often the mechanistic basis for such regulatory phenomena. This
makes enzymes such as deadenylases, which shorten poly(A) tails, and cytoplasmic poly(A)
polymerases (cPAP), which elongate poly(A) tails, important regulators of gene expression.
However, our understanding of these enzymes in multicellular systems is scarce. It is unclear in
the moment how the balance between polyadenylation and deadenylation contributes to the
development of a complex organ such as the germ line. We used the model organism C.
elegans to characterize the main cytoplasmic poly(A) polymerases (gld-2 and gld-4),
deadenylases (ccr-4, ccf-1, panl-2, parn-1, parn-2) and their interplay as one basis for the
functionality of germ cells. Using bulk poly(A) tail measurements we analyzed mRNA poly(A)
tails in the absence of specific deadenylases and cytoplasmic poly(A) polymerases. We found
that strong deadenylation defects were only detected in the absence of ccr-4 and ccf-1, which
are part of the Ccr4-Not complex. Interestingly, we detected strong polyadenylation defects in
the absence of gld-2 but not gld-4. Combined removal of gld-2 and ccr-4 or ccf-1 suggests
that a certain polyadenylation/deadenylation hierarchy exists in C. elegans. Currently, we are
testing if the removal of gld-2 or gld-4 together with specific deadenylases can rescue
cPAP-dependent germ line defects.
Page 210
Poster Abstracts
175
Understanding cotranslational protein folding at the molecular and cellular
levels using theory and computation
Edward O'Brien
University of Cambridge, United Kingdom
Presenter: Edward O'Brien
Understanding protein folding in living cells is one of the great challenges in biology. It requires
that we understand the concomitant folding of proteins with their biosynthesis by the ribosome
molecular machine, a factor shown to be important in determining the cellular concentration of
successfully folded proteins. I will discuss my recent efforts to understand the physical
principles of such cotranslational folding at the molecular and cellular scales through the
development of coarse-grained simulation force fields, chemical kinetic modeling,
bioinformatics techniques and systems biology methods. I will show how these tools have
allowed us to gain novel insights into fundamental issues of in vivo folding, including predicting
the impact of variable translation rates and synonymous codon usage [1], the molecular
mechanisms by which the chaperone trigger factor acts [2], and, at the cellular level, the
cotranslational folding properties of the E. coli [3] and S. cerevisiae [4] cytosolic proteomes.
These methods provide a quantitative theoretical framework for addressing key questions in
cellular nascent proteome behavior and are opening up new avenues of research in the areas
of synthetic biology, biomedicine and biotechnology.
References:
[1] E.P. O’Brien, M. Vendruscolo and C.M. Dobson. “Prediction of variable translation rate
effects on co-translational folding” Nature Communications , 2012 , 3, 868. [2] E.P. O’Brien, J.
Christodoulou, M. Vendruscolo and C.M. Dobson. “Trigger factor slows co-translational folding
through kinetic trapping while sterically protecting the nascent chain from aberrant cytosolic
interactions” J. Am. Chem. Soc. , 2012 , 134, 10920. [3] P. Ciryam, R.I. Morimoto, M.
Vendruscolo, C.M. Dobson and E.P. O’Brien. “In vivo translation rates can substantially delay
the co-translational folding of the E. coli cytosolic proteome” Proc. Natl. Acad. Sci. U.S.A. ,
2013 , 110, E132. [4] P. Ciryam, P. Sormanni and E.P. O’Brien. 2013 , In preparation.
Page 211
EMBO Conference Series: Protein Synthesis and Translational Control
176
Molecular insights into the effect of miRNAs on translation repression and
stimulation
Taran Limousin1, Ricardo Soto Rifo1, Emiliano Ricci1, Chloe Mengardi1, Paulina Rubilar1,
Theo Ohlmann2
1 INSERM, France
2 INSERM-ENS de LYON, France
Presenter: Theo Ohlmann
The mechanism by which microRNAs (miRNAs) can control gene expression has been a great
matter of debate. From the first studies in worm to the in vitro systems that are used today,
many models have been proposed that include regulation at the level of translation or at the
level of mRNA stability by controlling 3' deadenylation and decay. Recent studies provided a
consensus model of all these discrepancies and suggested that translation inhibition occured
first and is followed by deadenylation and further degradation of the target transcript.
Moreover, translation silencing seems to occur at the initiation level, and requires eIF4F and
PABP initiation factors. This led to the hypothesis that miRNAs could interfere with the
interaction between these two factors thus affecting the circularisation of the mRNA, which is
essential for translation efficiency. In order to gain insight into this mechanism, we have used
an in vitro system based on the rabbit reticulocyte lysate that fully recapitulates miRNA effects
on translation with virtually no effect on deadenylation and decay. Using this system and a
wide spectrum of translational inhibitors, we have narrowed down the step of initiation at which
repression is exerted and we found that miRNAs affect mainly ribosomal scanning. This effect
requires the presence of both eIF4G and PABP but does not rely on their physical interaction.
Further analysis of miRNA repression in cells revealed that the poly(A) tail was an absolut
requirement for miRNA action. To most of our surprise, we observed that removal of the
poly(A) resulted in a shift from repression to stimulation of mRNA expression. This effect seems
to require the middle domain of eIF4G and the presence of the Ago proteins. Altogether, these
results reveal the complexity of miRNA effect and open new prospects on translation
regulation.
Page 212
Poster Abstracts
177
Stimulation of ribosomal frameshifting by RNA G-quadruplex structures
Chien-Hung Yu1, Marie-Paule Teulade-Fichou2, René Olsthoorn1
1 Leiden University, The Netherlands
2 Université Paris-Sud, France
Presenter: René Olsthoorn
Guanine-rich sequences can fold into four-stranded structures of stacked guanine-tetrads,
so-called G-quadruplexes (G4). These unique motifs have been extensively studied on the
DNA level, however, exploration of the biological roles of G4s at the RNA level, is just
emerging. Here we show that G4 RNA when introduced within coding regions are capable of
stimulating -1 ribosomal frameshifting (-1 FS) in vitro and in cultured cells. Systematic
manipulation of the loop length between each G-tract revealed that the -1 FS efficiency
positively correlates with G4 stability. Addition of a G4-stabilizing ligand, PhenDC3, resulted in
higher -1 FS. Further, we demonstrated that the G4s can stimulate +1 FS and stop codon
readthrough as well. These results suggest a potentially novel translational gene regulation
mechanism mediated by G4 RNA.
Page 213
EMBO Conference Series: Protein Synthesis and Translational Control
178
Bacterial mRNA features affecting translation initiation and reinitiation – in vivo
and vitro studying
Ilya Osterman, Ekaterina Andreyanova, Maria Rubtsova, Elena Belova, Sergey Evfratov,
Alexey Bogdanov, Petr Sergiev, Olga Dontsova
Lomonosov Moscow State University, Russian Federation
Presenter: Ilya Osterman
Regulation of gene expression at the level of translation accounts for up to three orders of
magnitude in its efficiency. We systematically compared the impact of several mRNA features
on translation initiation at the first gene in an operon with those for the second gene. We have
found, that mRNA with extremely strong Shine Dalgarno (SD) sequence (8 nucleotides),
located very close to the start codon, is the most competitive, whereas for shorter SD (6
nucleotides) the optimal position is moved away from the start codon. We also systematically
checked the influence of the two hairpins (strong and weak) at the different positions on
translation efficiency. Hairpins located before SD don’t significantly affect translation, as
expected, while hairpins at the SD or start codon position strongly inhibit translation.
Interestingly strong hairpin after start codon inhibit translation, whereas weak hairpin increase
translation efficiency. We demonstrated significant differences in the efficiency of Shine
Dalgarno sequences acting at the leading gene and at the following genes in an operon. The
majority of frequent intercistronic arrangements possess medium SD dependence, medium
dependence on the preceding cistron translation and efficient stimulation by A/U-rich
sequences. The second cistron starting immediately after preceding cistron stop codon
displays unusually high dependence on the SD sequence. In addition to manual design of
UTRs, we made the library of the plasmids based on the dual fluorescent reporter plasmid
(CER/RFP) with randomized elements of 5’UTR (sequences between SD and AUG, before SD
or before AUG). E. coli cells were transformed with the set of randomized plasmids and sorted
according to the ratio of the CER and RFP fluorescence. Plasmids from the sorted cells were
sequenced and analyzed – this results revealed several conservative sequences, which
strongly affect bacterial translation.
Page 214
Poster Abstracts
179
Understanding the mechanism of differential regulation of insulin gene2 splice
variants
Poonam Pandey1, Amaresh Panda2
1 NCCS, India
2 NIH, United States of America
Presenter: Poonam Pandey
Understanding the insulin regulation is very important as this hormone plays a central role in
glucose metabolism. Mice express two insulin gene 2 splice variants termed mIns2L (L) and
mIns2S (S) that differ in their 5’UTR sequence without any change in the ORF. The differences
in the 5’UTR leads to different predicted secondary structures of these splice variants which
alters the binding of the trans-acting factors resulting in differential translation regulation. We
observed that the mIns2S is translated more efficiently and RNA EMSA suggests that factors
bind preferentially to the (L) indicating that these trans-acting factors are repressors. These
findings were also supported by the results from UVcross linking experiments. Further, our
studies indicate that PABP is one of the factors that bind differentially to these 5’UTR and
show that it binds to position 18 to 39 of the mIns2 5`UTR.This region is 50% ‘A’ rich and
contains 5 residues of ‘A’ nucleotide and shows no difference between both isoforms.The
secondary structure formed by the S does not allow effective binding of PABP to this region
while the secondary structure formed by the L isoform promotes the binding of PABP to this
region. However binding of PABP alone to the insulin 5`UTR does not lead to differential
translation suggesting that other factors may play a role in regulating the translation.Neuronal
specific transcription factor HuD was shown to bind to mINS2 5’UTR resulting in translation
repression and low insulin production (Lee et al 2011). RNA-EMSA experiments suggest that
binding complex contains both PABP and HuD. We also observed that HuD over expression
alone does not have any effect on translation regulation of both the splice variants. Also
immunoprecipitation assay confirmed that there is cross talk between PABP and HuD.
Identification of the mechanism of how these differentially binding factors regulate insulin gene
expression is being explored.
Page 215
EMBO Conference Series: Protein Synthesis and Translational Control
180
In vitro veritas: an adaptable mammalian cell free system that reproduces
physiological cellular conditions for in vitro translation
Baptiste Panthu, Didier Décimo, Laurent Balvay, Théophile Ohlmann
CIRI, Inserm U1111, Université Lyon 1, France
Presenter: Baptiste Panthu
In vitro systems have been widely used in protein production and in the studies of translational
control as they provide a fast, reproducible and easy to manipulate, way of studying gene
expression. However, one of the major draw back of these systems is that they generally
poorly reproduce physiological conditions found in living cells. Thus, we have designed and
engineered a novel mammalian protein expression cell free lysate (based on cell-free system)
highly effective which relies on the replacement of the ribosomes from the rabbit reticulocyte
lysate (RRL) with ribosomes isolated from any cultured cells of interest (Hela, Jurkat, BHK,
mouse stem cells, myoblasts, etc...) or from complete organs such as heart, brain, lung and
liver. This renders the system highly adaptable to different physiological conditions that
recapitulate faithfully translational characteristics found in the corresponding living cells such as
cap/poly(A) synergy, cell type specificity, tropism of IRES-driven translation and modifications
that can affect ribosomal structure. In addition, the adaptability of the hybrid system allows the
use of RNA interference to deplete any endogenous ribosome associated protein. Finally,
another key feature of this lysate is that it can be programmed either with synthetic RNAs or
natural RNAs that have been transcribed and processed in cellulo. We have used this latter
property to demonstrate, in vitro, the importance of the associated ribonucleoprotein complex
(RNP) for cytoplasmic gene expression. In summary, the major property of this novel system is
that it reproduces physiological conditions that are unique to living cells combined with the
adaptability and the high expression level of in vitro systems. This makes it an invaluable tool
for people working in the field of translational control.
Page 216
Poster Abstracts
181
The NS1 protein from influenza A is a general enhancer of the host translation
Baptiste Panthu1, Coralie Carron2, Didier Décimo1, Laurent Balvay1,
Manuel Rosa-Calatrava2, Théophile Ohlmann1
1 CIRI, Inserm U1111, Université Lyon 1, France
2 VirPath EMR 4610, Université de Lyon, Université Claude Bernard Lyon 1, Hospices Civils
de Lyon, Faculté de médecine RTH Laennec, France
Presenter: Baptiste Panthu
The multifunctional nonstructural protein NS1 of influenza A viruses acts as a key modulator on
the global pathway of the host infected cells including the hijacking of the host
post-transcriptional processes. As such, it was shown to be involved both in the control of viral
and cellular translation; however the mechanism by which it acts remains rather controversial
and need to be further deciphered. Thus, we have revisited the role of NS1 in translation by
using a combination of influenza infection together with RNA transfection of reporter genes and
in vitro translational assays. Our data show that the NS1 protein is able to enhance translation
of virtually all tested mRNAs including those from influenza and genes whose expression was
driven from a wide spectrum of cellular and viral IRESes including picornavirus and hepatitis C.
Interestingly, the only RNA that was resistant to NS1 stimulation was that derived from the
CrPV genomic RNA. Further investigation into the molecular mechanism revealed that the
amino-terminal RNA binding region of NS1 is required for this translation stimulation and
preliminary data suggest that NS1 may interact directly with the multi subunit initiation factor
eIF3. A model for translation enhancement by the NS1 protein is proposed where the latter can
tether to mRNAs to directly bind the 43S ribosomal subunit.
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EMBO Conference Series: Protein Synthesis and Translational Control
182
Maintaining fidelity? A glimpse into the rules governing mammalian start codon
selection
Lisa Perry, Joanne Cowan, Harvey Johnston, Lucinda Eaton, Samantha Hodges, Kevin
Jones, Mark Coldwell
University of Southampton, United Kingdom
Presenter: Lisa Perry
The original scanning model of start codon selection states that translation initiation must
occur at the first AUG in a strong context and alternative routes are considered a loss of
fidelity. But, alternative initiation of translation is emerging as an increasingly understood source
for diversifying genome expression. This includes utilisation of multiple start sites, non-AUG
codons and IRESs that contribute to the generation of multiple isoforms of proteins that may
show differences, for example, in sub-cellular localisation and function. Deregulation of
alternative initiation is establishing itself as a major factor in disease; cancer, Alzheimer’s and
motor neurone disease. Past research has focused on understanding mechanisms responsible
for translation fidelity in eukaryotic systems, but the impact of alternative initiation has not been
considered. We describe the ‘rules’ governing start codon selection. This has been achieved
by exploring the importance of cis -acting elements, such as: start codon, context and
secondary structure; and manipulation of several trans-acting factors including eukaryotic
initiation factors in human tissue culture cell lines. To ensure applicability, we explored ‘real’
mRNAs already shown to use alternative initiation. In addition to this, we show evidence of
novel candidates using alternative initiation.
Page 218
Poster Abstracts
183
Cytoplasmic polypyrimidine tract binding protein controls mRNAs that regulate
cytoskeletal organisation and cell migration
Xavier Pichon1, Mark Stoneley1, Kirsty Sawicka1, Ruth Spriggs1, Laura Cobbold1, Keith
Spriggs2, Martin Bushell1, Anne Willis1
1 MRC Toxicology Unit, United Kingdom
2 University of Nottingham, United Kingdom
Presenter: Xavier Pichon
Polypyrimidine tract binding protein (PTB) and its neuronal paralog (nPTB) are multifunctional
RNA binding proteins that participate in various post-transcriptional mechanisms of gene
expression, including pre-mRNA splicing, mRNA stability, mRNA localization, and mRNA
translation. By combining two microarray-based post-transcriptional screens we show that
approximately 25% of the mRNAs that PTB/nPTB regulates in the cytoplasm encode proteins
involved in cytoskeletal signalling, cell migration and actin polymerisation. We demonstrate that
PTB/nPTB controls both the translation and the localization of these mRNAs through
sequence elements in their 3’UTRs. In support of role for PTB/nPTB in regulating the
cytoskeleton we show that siRNA-mediated depletion of these proteins has a dramatic effect
on cell morphology, stress fibre formation and cell migration. Our data defines a novel pathway
through which cytoplasmic PTB controls cytosketetal organisation and cell migration
Page 219
EMBO Conference Series: Protein Synthesis and Translational Control
184
Do EJCs enhance L-myc IRES dependent translation?
David Piñeiro1, Tuija Pöyry1, Christian M Lucas2, Ian C Eperon2, Anne Willis1
1 MRC, Toxicology Unit, United Kingdom
2 University of Leicester, United Kingdom
Presenter: David Piñeiro
Internal ribosome entry sites (IRES) are complex RNA structural elements that are located in 5'
untranslated regions (5'UTR) and they act to regulate protein syntheses from these mRNAs.
IRESs were first identified in picornaviruses, but our lab and others have shown that many
cellular mRNAs also use these elements to initiate translation. The 5’UTRs of the mRNAs
transcribed from the proto-oncogene myc family genes (N-, L-, c-myc) contain IRESs, which
allow them to be translated in an IRES-dependent manner under cellular stress. All members
of this family also contain three exons and two introns in theirs pre-mRNAs. The main initiation
codon is located toward the 5’ end of exon 2. It has been suggested that the exon junction
complexes (EJCs) of the spliced mRNAs enhances translation. The EJC is a multiprotein
complex comprising at least Magoh, Y14, eIF4AIII and BTZ, which is deposited 20-24 nt
upstream of the exon-exon junctions as a result of splicing. The EJC is a major constituent of
spliced mRNPs and moves with the mRNA from the nucleus to the cytosol where it is
disassembled during the ‘pioneer’ round of translation. The EJCs are also involved in
surveillance and exportation. Within cells there are two L-myc mRNAs which differ in their
5’UTR lengths. The short version of the mRNA (sL-myc) contains a functional IRES, whereas
the long version (lL-myc) contains an intron A within the IRES element. When the lL-myc mRNA
is spliced, there will be an EJC within the IRES element and this could have an effect on
IRES-driven translation. To explore this possibility, we designed 4 constructs with different
5’-UTRs: globin (intronless), lL-myc, sL-myc and sLmyc+globin intron, followed by a luciferase
reporter. All transcripts were translated in RRL assay before and after being spliced. The
plasmids were also transfected in Hela cells to test the translation efficiency. These data could
shed light on a new function of the EJCs in the IRES-dependent translation.
Page 220
Poster Abstracts
185
eIF4E-mediated translational control of the stem/progenitor cell transition to
malignancy: implications for pregnancy-associated breast oncogenesis
Svetlana Avdulov, Jeremy Herrera, Petersen Mark, Karen Smith, Jose Gomez-Garcia,
Matthew Parker, Thomas Beadnell, J. Carlos Manivel, Kaylee Schwertfeger, Douglas Yee,
Peter Bitterman, Vitaly Polunovsky
University of Minnesota, United States of America
Presenter: Vitaly Polunovsky
Pathological eIF4E function is a hallmark of established malignancies. In most tumors, temporal
deregulation of eIF4E-mediated translation is due to sustained upstream signals emanating
from activated oncogenes. Whether deregulation of the translational apparatus is required for
oncogenesis, or whether increased translational rates per se are sufficient for the genesis of
cancer remains undefined. Pregnancy is accompanied by activation of the protein synthesis
machinery for an extended period of time. Parous women experience a poorly understood
increased risk of breast cancer soon after delivery. To determine whether hyperactivation of
eIF4E-driven translation suffices to promote pregnancy-associated breast cancer, we
developed a transgenic mouse model in which the whey acidic protein (WAP) promoter directs
reversible expression of eIF4E to mammary stem/progenitor cells (MaSPCs). In this model
system, exogenous eIF4E is turned “ON” by lactogenic hormones during late pregnancy and
lactation and is switched “OFF” by weaning. Pregnancy-associated over expression of eIF4E
promoted hyper-proliferation of MaSPCs and resulted in formation of multiple neoplastic foci
culminating in invasive cancer. When exogenous eIF4E was silenced by weaning after
neoplastic foci had already formed, progression to frank tumors did not occur. A genome-wide
analysis of the transcriptome and translatome showed that acquisition of eIF4E-induced
neoplastic lesions was associated with altered translational control of genes assigned to the
translation and cell adhesion ontologies. These findings indicate that distinct threshold levels of
eIF4E govern normal proliferation and oncogenic expansion of stem/progenitor cells and that
levels of eIF4E above the pro-neoplastic threshold can be a primary event in oncogenesis.
They also show that increased rates of translation during pregnancy create a high-risk state in
which relatively subtle changes in eIF4E-mediated translation may trigger oncogenesis.
Page 221
EMBO Conference Series: Protein Synthesis and Translational Control
186
RBP atlas: an exploration of interactions between mRNA and proteins and
their impact on cardiomyocyte biology
Yalin Liao1, Alfredo Castello2, Sophia Foehr2, Stefan Leicht2, Bernd Fischer2, Rastislav
Horos2, Jeroen Krijgsveld2, Matthias Hentze2, Thomas Preiss1
1 The Australian National University, Australia
2 EMBL Heidelberg, Germany
Presenter: Thomas Preiss
RNA-binding proteins (RBPs) control all aspects of RNA fate and defects in their function
underlie a broad spectrum of human pathologies. Employing a combination of UV-crosslinking
of proteins to RNA in living cells with identification of those co-purifying with poly(A)+ RNA by
mass spectrometry, we recently identified 860 proteins as the “mRNA interactome” of human
HeLa cells. Over 300 of these RBPs were not previously known to bind RNA and their
identification sheds new light on RBPs in disease, RNA-binding enzymes of intermediary
metabolism, RNA-binding kinases, and RNA-binding architectures. We have now adapted this
approach to identify the mRNA interactome of murine HL-1 cardiomyocytes. The HL-1 cell line
maintains the ability to contract and other differentiated cardiac morphological and functional
properties in culture. Our analyses reveal both, RBPs commonly detected in cells of different
origin as well as more cardiomyocyte-specific RBPs. Ongoing work is focused on detecting
changes in the cardiomyocyte mRNA interactome under conditions of pathophysiological
stress and on identifying the RNA targets of selected cardiomyocyte RBPs using RBP
pull-down followed by next generation sequencing of co-purifying RNA. Altogether, these
investigations will map networks of post-transcriptional gene regulation in cardiomyocytes and
might reveal their involvement in molecular processes commonly at play in heart disease.
Page 222
Poster Abstracts
187
miRNA profiles characterise distinct states of cellular pluripotency
Jennifer Clancy1, Hardip Patel1, Nicole Cloonan2, Andrew Corso3, Mira Puri3, Pete Tonge3,
Andras Nagy3, Thomas Preiss1
1 The Australian National University, Australia
2 Queensland Institute of Medical Research, Australia
3 Samuel Lunenfeld Research Institute, Canada
Presenter: Thomas Preiss
Reprogramming of fibroblasts through induction of the Yamanaka factors (Myc, Sox2, Klf4 and
Oct4) is a powerful approach to delineate the molecular characteristics of the pluripotent
cellular state. We used an efficient secondary reprogramming system to monitor whole culture
reprogramming over time, allowing us to characterise intermediate states as well as to identify
several distinct pluripotent cell states. As part of this investigation we measured the miRNA
profiles of these cell states by next-generation sequencing, which feature marked changes in
the expression of many of the previously described core miRNAmediators of pluripotency. We
uncover the timing of their involvement in the process of pluripotent cell generation, which
sheds light on their individual roles in the process, as well as on how miRNAs are regulated
during reprogramming. We describe the miRNA processing variants that exist in these cell
states, including 5’ and 3’ isomiRs, non-templated addition, editing and unusual strand bias.
Many of the core reprogramming miRNAs have isomiRs, which can alter their targeting
spectrum and lead to reinterpretation of their specific roles. Broader analysis of the dataset
also suggests that other small RNA species may be involved in pluripotency as, like the miRNA
population itself, the small RNA profile changes markedly after induction of the Yamanaka
factors. This work is part of a larger collaborative study, which co-ordinately measured protein,
miRNA, mRNA, DNA methylation and histone modification in this model of pluripotent cell
generation and related data will be discussed in the context of miRNA regulation and function.
Page 223
EMBO Conference Series: Protein Synthesis and Translational Control
188
Shifting targets: microRNA variants and alternative polyadenylation in cardiac
hypertrophy
Carly Hynes1, David Humphreys2, Nicola Smith2, Hardip Patel1, Robert Graham2, Jennifer
Clancy1, Thomas Preiss1
1 The Australian National University, Australia
2 Victor Chang Cardiac Research Institute, Australia
Presenter: Thomas Preiss
Emerging findings indicate that cells can produce both miRNAs and their mRNA targets in
multiple processing variants as a means to increase the complexity of miRNA-mediated control
in a tissue and developmental stage-specific manner. MiRNAs play critical roles in the heart,
and we hypothesise that during the cardiac hypertrophic response there are changes to both
miRNA processing and mRNA 3’ polyadenylation site selection, which will alter miRNA/mRNA
interactions. Transverse Aortic Constriction (TAC), a model of left ventricular hypertrophy, was
used to obtain pre-hypertrophic and hypertrophic cardiomyocytes. TAC led to pressure
overload and left ventricular hypertrophy, characterised by an increase in left ventricular weight
and induction of hypertrophic markers. RNA was then extracted from purified cardiomyocytes
for next-generation sequencing of small RNAs and mRNA 3’ ends. Several miRNAs were
deregulated prior to the development of hypertrophy and in the hypertrophic hearts.
Processing variants, such as isomiRs and unexpected arm bias, of key cardiac miRNAs were
also identified that could potentially alter their targeting specificity. Furthermore, numerous
mRNAs encoding important cardiac functions are subjected to alternative polyadenylation that
alters the length of the 3’UTR. The 3’UTR length changes may alter the extent to which
miRNAs can regulate these mRNAs. The sequencing has produced global information on
expression changes to both miRNA sequence and mRNA 3’UTR lengths, allowing us to form a
systems level understanding of miRNA-regulation during cardiac hypertrophy. The realisation
that cardiac miRNAs and their targets exist as currently under-appreciated variants with
potentially complex effects on target specificities has important implications for the role of
miRNAs in cardiac disease.
Page 224
Poster Abstracts
189
A tale of two termini: profiling mRNA 5'-3' interactions in vivo
Stuart Archer, Thomas Preiss
The Australian National University, Australia
Presenter: Thomas Preiss
Interactions between the 5' and 3' termini of mRNAs have long been postulated to occur in the
cell. This "closed-loop" model of mRNA is attractive from a number of viewpoints: functionally,
the closed loop explains the ability of 3' UTR features to modulate events occurring at the 5'
end such as translation initiation, while mechanistically, the 5' cap structure is known to bind
the eIF4F complex, which also interacts with the poly(A)-tail via PABP, conceptually forming a
closed loop mRNP if all interactions occur simultaneously. Here we introduce a novel assay to
detect the closed-loop conformation of specific mRNAs in vivo for the first time. Using
Saccharomyces cerevisiae, we demonstrate that the closed-loop is the predominant
conformation found in mRNAs bound by eIF4F, but also introduce the possibility that this
structure is not adopted by all mRNAs during normal growth. While the closed-loop
conformation has been reconstituted in vitro by mixing eIF4F and PABP with mRNA, the
situation may be different in the cell. Various in vivo examples are known where interactions
between the eIF4F subunits and PABP come under regulatory intervention, either globally or in
a transcript-specific manner, resulting in reduced translational efficiency. Thus, detecting
altered closed-loop status represents a catch-all method of identifying whether mRNA is under
translational regulation. We are investigating the global and transcript-specific regulation of the
closed-loop conformation in response to environmental cues, which will identify regulatory
mechanisms that have been expanded upon during the evolution of more complex organisms
to rapidly fine-tune gene expression.
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EMBO Conference Series: Protein Synthesis and Translational Control
190
RNA methylation: a mechanism for post-transcriptional regulation that is
deregulated in cancer?
Tennille Sibbritt1, Brian Parker1, Hardip Patel1, David Humphreys2, Jeffrey Squires2, Susan
Clark3, Thomas Preiss1
1 The Australian National University, Australia
2 Victor Chang Cardiac Research Institute, Australia
3 Garvan Institute of Medical Research, Australia
Presenter: Thomas Preiss
Internal methylation of eukaryotic RNA in the form of N6-methyladenosine (m 6 A) and
5-methylcytosine (m 5 C) have been known to exist for decades, however, laborious detection
methods have limited the understanding of their role. With the availability of high-throughput
sequencing techniques, these drawbacks have been overcome, revealing non-random
distribution of internal methylation in a wide variety of RNA biotypes. Early investigation into the
prevalence of 5-methylcytosine (m 5 C) in RNA has largely been confined to tRNA and rRNA.
Recently, we implemented a bisulfite sequencing-based technique for transcriptome-wide as
well as locus-specific detection of m 5 C and mapped thousands of m 5 C sites in the human
transcriptome including in mRNA and non-coding RNA. Biased distribution of m 5 C between
and within mRNAs, e.g. enrichment in the untranslated regions, is consistent with roles in
post-transcriptional regulation of gene expression. m 5 C is a key DNA modification associated
with epigenetic gene regulation in mammalian cells and is also known to be deregulated in
cancer. We have now begun to investigate the enzymes responsible for modifying mRNA by
an RNAi approach as well as a role of RNA methylation in cancer by comparing the m 5 C
profiles of normal prostate cells (PrEC) and metastatic prostate cancer cells (LNCaP). Analysis
of the recorded patterns of m 5 C sites in mRNA shows many transcripts are differentially
methylated between each cell line. Currently, we are consolidating and extending the potential
link of m 5 C to post-transcriptional regulation and cancer, as well as addressing the molecular
function of methylation in mRNA.
Page 226
Poster Abstracts
191
A novel high throughput biochemical assay to evaluate HuR-RNA complex
formation
Vito D'Agostino1, Marialaura Amadio2, Christopher Tiedje3, Valentina Adami1, Matthias
Gaestel3, Alessandro Provenzani1
1 CIBIO, University of Trento, Italy
2 University of Pavia, Italy
3 Hannover Medical School, Germany
Presenter: Alessandro Provenzani
The RNA-binding protein HuR/ELAVL1 binds to AU-rich elements (AREs) promoting the
stabilization and translation of a number of mRNAs into the cytoplasm, dictating their fate. We
applied the sensitive AlphaScreen technology using purified human HuR protein, expressed in
a mammalian cell-based system, to characterize its binding performance in vitro towards a
ssRNA probe corresponding to the AREs of TNFα 3’UnTranslated Region. We analyzed the
binding kinetics in saturation and time course experiments, including competition assays. The
method revealed to be a successful tool for determination of HuR binding kinetic parameters in
the nanomolar range, with calculated Kd of 2.5±0.60 nM, kon of 2.76*106±0.56 M-1 min-1,
and koff of 0.007±0.005 min-1. We also tested the HuR-RNA complex formation by
fluorescent probe-based RNA-EMSA. Moreover, we adapted the AlphaScreen assay in 384well plate format and obtained a Z-factor of 0.84 and an averaged coefficient of variation
between controls of 8%, indicating that this biochemical assay fulfills criteria of robustness for a
targeted screening approach. By screening 2000 molecules we identify one compound (D1)
that, in breast cancer MCF-7 cells, decreases the level of secreted TNFα protein with a
concomitant reduction of TNFα mRNA polysomal loading. Interestingly, the expression of total
TNFα mRNA does not change upon treatment, suggesting a post-transcriptional mechanism
of action of D1. Consistently with our in vitro data, the number of TNFα mRNA copies is lower
in HuR immunoprecipitates of treated samples compared to the controls. Summarizing, we
show here a method to identify small molecules able to interfere with HuR binding to its
cognate mRNAs both in vitro and in cells. Moreover given its versatility, this tool could be
applied to other RNA-Binding Proteins recognizing different RNA, DNA, or protein species,
opening new perspectives in the identification of small-molecule modulators of RNA binding
proteins activity.
Page 227
EMBO Conference Series: Protein Synthesis and Translational Control
192
Exploring the role of Dhh1-Ribosome interactions in general translation
repression
Aditya Radhakrishnan, Rachel Green
Johns Hopkins Medical Institute, United States of America
Presenter: Aditya Radhakrishnan
The yeast protein Dhh1 has long been implicated in regulation of translation by promoting
mRNA decapping [1] as well a more general repression of translation of mRNA transcripts
through a cap-independent mechanism [2]. Interestingly, when Dhh1 is tethered to the 3’ UTR
of a message, the targeted mRNA becomes fully loaded with ribosomes and sediments very
deep in a sucrose gradient; these studies suggest that Dhh1 might specifically hinder slow
ribosomes [3]. Broadly speaking, these results suggest the possibility of a direct interaction
between Dhh1 and the ribosome. The existence and nature of this interaction has been
queried using a combination of in vitro biochemical assays using recombinantly expressed
Dhh1 along with in vivo polysome profile analysis. To aid in our polysome profiling, we have
designed a set of reporters to monitor not only the presence, but also positioning, of
ribosomes along reporter mRNA transcripts in response to varied levels of Dhh1 in the cell.
Through these studies, we hope to shed light onto the interaction between Dhh1 and the
ribosome, and its role in general translation repression.
References:
[1] Fischer N, Weis K EMBO J 2002. 21: 2788-2797 [2] Coller J, Parker R Cell 2005. 122:
875886 [3] Sweet T, Kovalak C, Coller J PLOS Biology 2012. 10 (6): 1-15
Page 228
Poster Abstracts
193
Homestatic plasticity - paradigm to study regulation of protein translation by
microRNAs
Marek Rajman1, Marcus Krüger2, Thomas Braun2, Gerhard Schratt1
1 Institute of Physiological Chemistry, University of Marburg, Marburg, Germany
2 Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
Presenter: Marek Rajman
Individual neurons and neuronal networks must retain stable function in the face of many
destabilizing events which occur during neural circuit development, learning and memory
formation. Homeostatic plasticity mechanisms that keep neuronal activity within a narrow
range are used by neurons to counterbalance these destabilizing influences. Dynamic turnover
of proteins is required for homeostatic plasticity, but little is known regarding the specific nature
of these proteins. In the present study, we used SILAC connected to mass spectrometry (MS)
to characterize changes in new protein synthesis in synaptic scaling (specific form of
homeostatic plasticity). We chronically increased network activity by applying the GABA-A
receptor blocker picrotoxin (PTX) for 48h to 18 days in vitro (DIV) rat hippocampal neuron
cultures (HC) devoid of glial cells. We showed that neuron enriched HC were able to
downscale (↓ of surface Glur1 (60%), Glur2 receptors (20%)) after PTX application and could
therefore be used to study synaptic scaling. We applied pulsed SILAC (Arg6/Lys4) to
differentiate newly synthesized proteins from pre-existing ones. The following experimental
setup was used: 14DIV – application of Arg6/Lys4; 18DIV – application of PTX; 20DIV –
extraction of the proteome from control and PTX samples, mixed 1:1 with Spike-in standard
(Arg10/Lys8) followed by MS analysis. Results from a pilot screen (comparing 1189 proteins)
showed a positive correlation between changes in the levels of new and old proteins for only
39% of all proteins. This suggests that for at least 60% of the analyzed proteins, we could
successfully distinguish between new synthesis and stability of pre-existing protein. Moreover,
we found first candidate proteins which show changes in new synthesis upon PTX treatment.
In the future, we plan to extend our analysis to more comprehensive MS screens and study the
involvement of microRNAs in the regulation of new protein synthesis during synaptic scaling.
Page 229
EMBO Conference Series: Protein Synthesis and Translational Control
194
Characterisation of novel methyltransferases involved in modifications of
mitochondrial large subunit rRNA
Joanna Rorbach, Pierre Boesch, Payam Gammage, Michal Minczuk
MRC MBU, United Kingdom
Presenter: Joanna Rorbach
Mitochondria possess a genome that encodes essential subunits of the oxidative
phosphorylation system. Defects in mitochondrial gene expression have been linked with many
neurodegenerative disorders and the ageing process. Despite the critical importance of
mitochondrial gene expression for cell function, it is surprising how little is known about its
details. This is particularly true for the processes underlying post-transcriptional control and
mitochondrial translation. The mitochondrial 55S ribosome is composed of a 39S large subunit
(mt-LSU) and a 28S small subunit (mt-SSU), containing the 16S and 12S rRNA, respectively.
Maturation of rRNA requires a number of post-transcriptional nucleotide modifications, mostly
base methylation, 2′-O-ribose methylation and pseudouridylation. Not much is known about
maturation of the human mitochondrial 16S rRNA and its role in biogenesis of the
mitoribosome. Currently, mTERF4-NSUN4 is the only recognised complex that binds 16S
rRNA, although its function as 16S rRNA-specific methyltransferase needs to be validated in
vivo [1]. Here, we characterise two novel methyltransferases (MRM2 and MRM3) that are
responsible for modifications in the nucleotides of the 16S rRNA A-loop, an essential
component of the peptidyl transferase centre. Our studies show that inactivation of MRM2 or
MRM3 in human cells by RNA interference results in respiratory incompetence owing to
reduced mitochondrial translation rates. Ineffective translation in MRM2 and MRM3-depleted
cells results from aberrant assembly of mt-LSU. These findings lead us to propose that MRM2
and MRM3 are novel human mitochondrial methyltransferases involved in modification of 16S
rRNA and are important factors for the biogenesis of the large subunit of the mitochondrial
ribosome.
References:
[1] MTERF4 regulates translation by targeting the methyltransferase NSUN4 to the mammalian
mitochondrial ribosome. Camara Y. et al. (2011) Cell Metab.13:527–539.
Page 230
Poster Abstracts
195
eIF5A binds directly to the 60S ribosomal subunit
Danuza Rossi1, Christopher Fraser2, John Hershey2, Sandro Roberto Valentini1, Cleslei
Zanelli1
1 UNESP, Brazil
2 University of California, United States of America
Presenter: Danuza Rossi
The putative translation initiation factor 5A (eIF5A) is essential for cell viability and highly
conserved in archaea and eukaryotes. Initially purified from ribosomes of reticulocyte lysates,
eIF5A was shown to stimulate the formation of the first peptide bond, and has also been
shown to play a role in elongation. Active eIF5A contains an unusual and essential
post-translational modification generating the hypusine residue (Hyp). To better understand the
role of eIF5A in the control of protein synthesis, we investigated the binding of eIF5A to the
ribosome using fluorescence anisotropy. Since this strategy requires the use of single-cysteine
on the surface of the query protein, we generated six functional single-cysteine versions of
human eIF5A. Production of active eIF5A Hyp was achieved using a polycistronic system in E.
coli, co-expressing the two enzymes of the hypusine-modification pathway. We also purified
ribosomes (80S) and ribosomal subunits 60S and 40S. Cell-purified pre-assembled 80S
ribosomes containing endogenous eIF5A were used in a competition assay to demonstrate
that recombinant eIF5A is also functional in vitro. Interestingly, eIF5A Hyp competes with
endogenous eIF5A on pre-assembled 80S ribosomes much more efficiently than the
unmodified eIF5A. The fluorescence anisotropy data obtained from eIF5A Hyp -60S complex
formation showed approximate K d =200 nM value. These results demonstrate the binding of
eIF5A to the 60S ribosomal subunit and will serve as a base for subsequent mapping of the
site of eIF5A binding in the ribosome. Supported by FAPESP, CAPES and CNPq.
Page 231
EMBO Conference Series: Protein Synthesis and Translational Control
196
NSUN4 is a bi-functional methyltranferase required for the biogenesis of the
mitochondrial ribosome
Benedetta Ruzzenente1, Metodi Metodiev2, Yolanda Camara3, Chan Bae Park4, Henrik
Spåhr1, Paola Loguercio Polosa5, Caroline Meharg6, Christian Becker7, Janine Altmueller7,
Bianca Habermann1, Nils-Göran Larsson1
1 Max-Planck Institute for Biology of Ageing, Germany
2 Hôpital Necker-Enfants Malades, France
3 Hospital Universitari Vall d0Hebron, Spain
4 Ajou University School of Medicine, Republic of Korea
5 University of Bari, Italy
6 Queen's University, Ireland
7 Cologne Center for Genomics, University of Cologne, Germany
Presenter: Benedetta Ruzzenente
Mitochondria perform a number of essential functions in the cell including synthesis of ATP via
the oxidative phosphorylation (OXPHOS) system. Normal mitochondrial function requires
coordinated expression of two genomes: mitochondria’s own genome (mtDNA), which
encodes 13 respiratory chain subunits with essential structural and functional role for the
OXPHOS system, and the nuclear genome encoding the remaining ~ 80 subunits. The
mtDNA-encoded polypeptides are synthesized on mitochondrial ribosomes (mitoribosomes)
located in the mitochondrial matrix. Biogenesis, maintenance and regulation of the complex
translation apparatus are essential for mitochondrial function. Using conditional knockout
mouse models, we recently characterized two proteins involved in the biogenesis of the
mitochondrial ribosome: the m 5 C-methyltransferase NSUN4 and its interaction partner the
RNA-binding protein MTERF4. Both genes are essential for embryonic development and
tissue-specific disruption of either Mterf4 or Nsun4 in the heart causes cardiomyopathy with
mitochondrial dysfunction. Previous studies proposed that the MTERF4-NSUN4 complex
regulates ribosomal assembly by methylation of an unknown residue in the mitochondrial 16S
rRNA. Using deep sequencing of RNA, isolated from control and Nsun4 -mutant mice we
identified the methylation site for NSUN4. Interestingly, comparison of the rRNA methylation
status in both, Nsun4 - and Mterf4 -mutant mice showed that, unlike previously thought,
interaction between NSUN4 and MTERF4 is dispensable for methylation of the NSUN4 target
substrate. The NSUN4-MTERF4 complex itself plays an essential role for monosome formation
which is independent of the methyltransferase activity of NSUN4. We propose that NSUN4 is a
bi-functional protein, which on one hand is needed for rRNA methylation and, on the other
hand, in complex with MTERF4, plays an important role in monosome formation possibly by
preventing premature assembly of both mitoribosomal subunits.
Page 232
Poster Abstracts
197
Conserved non-AUG translation initiation in the mRNA of cpc-1 from
Neurospora crassa suggests potential for previously unrecognised layer of
regulation
Ivaylo Ivanov1, Jiajie Wei2, John Atkins3, Matthew Sachs2
1 University College Cork, Ireland
2 Texas A&M University, United States of America
3 University of Utah/University College Cork, United States of America
Presenter: Ivaylo Ivanov
Neurospora crassa cross-pathway control 1 (cpc-1) and Saccharomyces cerevisiae GCN4 are
homologs specifying a transcription activator, which drives the primary transcriptional response
to amino acid starvation. The translation of upstream open reading frames (uORFs) controls
the expression of GCN4. GCN4 uORF1 is constitutively translated and promotes reinitiation
either at the start codon of GCN4 in response to amino acid limitation or at the start codon of
uORF4 under non-limiting conditions to inhibit the translation of GCN4. Previously, it was
shown that cpc-1 mRNA contains two uORFs and its expression is controlled at the level of
translation in response to amino acid starvation. We used cell-free extracts to show that cpc-1
uORF1 and uORF2 are functionally analogous to uORF1 and uORF4 in GCN4. We also report
an unexpected finding that the 5’ region upstream of the main coding sequence of the cpc-1
mRNA contains another feature likely important for the translation regulation of cpc-1. All
filamentous fungi containing cpc-1 homologs, for which sequence data are available (from 100
different species), have a potential 5’ extension of the cpc-1 coding region that precedes the
uORF2 start codon. We identified four near-cognate non-AUG codons, some showing deep
conservation, in the cpc-1 mRNA zero frame and upstream of uORF2. These codons can be
used for translation initiation in N. crassa though with a reduced level in vivo than in vitro. We
speculate that initiation events at non-canonical codons, which bypass the inhibitory uORF2,
could be used as a sensor for activating cpc-1 expression in response to physiological
conditions that reduce the stringency of start codon selection. Ribosome profiling provided
further information about the ribosome distribution on the cpc-1 mRNA.
Page 233
EMBO Conference Series: Protein Synthesis and Translational Control
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Visualisation of +1 frameshifting during tRNA accommodation
Matthieu Saguy1, C. Alistair Siebert2, Hélène Chommy1, David Cornu3, Olivier Namy1, Robert
J. C. Gilbert2
1 Université Paris Sud, France
2 Wellcome Trust Centre for Human Genetics, Univeristy of Oxford, United Kingdom
3 Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif, CNRS, France
Presenter: Matthieu Saguy
At the level of frameshift site, ribosomes are able to change translation frame in a –1 or +1
direction. The frameshift sites are composed of a slippery sequence and a structured
stimulatory element to the 3’ of the slippery sequence, such as a stem-loop or pseudoknot.
Here we describe the structural analysis of ribosomes programmed with the +1 frameshifting
mRNA for the mammalian ornithine decarboxylase antizyme 1 (OAZ1). We first show that the
OAZ1 frameshift is associated with a pause in translation elongation; determining the
structures of ribosomes from the paused reaction by cryo-electron microscopy and single
particle reconstruction reveals two different conformational states. In one state, 40S domain
closure has not occurred so that the ribosomal A-site remains open, but the translation
elongation factor eEF1A is bound and the framseshifting pseudoknot is apparent. In the other,
the ribosome adopts an A-site closed state and contains a P-site tRNA and no apparent
pseudoknot structure. We confirm the identity of the bound eEF1A using mass spectrometry
and show that it is located similarly to EF-Tu-tRNA in prokaryotic ribosomes, but not
identically. We conclude that the presence of eEF1A identifies a pre-frameshift ribosomal state,
while the P-site tRNA-bound state is post-frameshift. Thus, antizyme-associated +1
frameshifting occurs by the out-of-frame engagement of a tRNA at the A-site. While +1
frameshifting has evolved at the accommodation stage of translation elongation, we previously
showed that -1 frameshifting occurs at the translocation stage.
Page 234
Poster Abstracts
199
High content screening approach to identify translational regulators of RNA
transport
Anca Savulescu, Robyn Brackin, Musa Mhlanga
CSIR, South Africa
Presenter: Anca Savulescu
Subcellular localization of mRNA, followed by the spatial regulation of translation is an
evolutionary conserved mechanism to achieve the precise control of protein synthesis in
sub-cellular locations. The spatial segregation of mRNAs within cells has been observed in a
wide variety of cell types, including fibroblasts, neurons, oocytes and zygotes. A commonality
of all long-range mRNA transport is translational repression until completion of the transport
process and triggering of protein synthesis. To elucidate the mechanisms of mRNA transport
and translational control that are shared between multiple mRNAs, we combined fluorescence
microscopy customized image analysis of cell shape with biochemical assays. Migrating
mouse fibroblasts contain mRNAs that are enriched in the pseudopodia upon stimulation with
various agents. We applied single molecule FISH in order to visualize localized mRNAs, as well
as immunofluorescence to track the proteins translated from these localized mRNA at differing
time points in the transport process. The localization relative to the cell shape was then
calculated using a bespoke image analysis algorithm, which identifies the centers and edges of
individual cells. The biochemical assay consists of pull-down experiments using biotinylated
probes against localized mRNAs, followed by mass-spectrometry analysis of associated
proteins. We identified a list of proteins associated with several localized mRNAs, which we
further categorized into sub compartments by applying similar pull-downs from nuclear and
cytoplasmic cellular fractions. By comparing proteins associated with different localized
mRNAs, we hypothesized that they may be evolutionarily conserved at different stages in the
hierarchical mRNA transport process. The assay and data were validated using siRNA
techniques and western blot analysis revealing novel and broadly conserved mechanisms of
mRNA regulation during its transport.
Page 235
EMBO Conference Series: Protein Synthesis and Translational Control
200
Characterization of Yeast eIF4E post-translational modification
Manisha Saxena, Michael Altmann
University of Bern, Switzerland
Presenter: Manisha Saxena
Eukaryotic initiation factor 4E (eIF4E), cap-binding protein, participates in recruitment of mRNA
to the ribosome during translation initiation. Most eukaryotic organisms express multiple
eIF4E’s for general translation and/or for specialized functions like repression of translation. In
this study we used in vivo phenotypic, electrophoretic and spectrometric analysis to map
posttranslational modifications of yeast Saccharomyces cerevisiae eIF4E mutants. In vivo
phenotypic examinations included temperature sensitivity, adherence ability to solid surfaces
and m7GTP binding affinity. Spectrometric analysis revealed amidation at C-terminus and
potential phosphorylation and deamidation in the unstructured N-terminus of eIF4E containing
series of serine and threonine residues. C-terminal truncated eIF4E mutants showed reduction
to two isoforms when compared to 4EWt on 2D-blots and reduced cap-binding affinity.
Interestingly, deletion of amino acids in N-terminus resulted in in vivo phenotype and several
isoforms on 2D-electrophoretic analysis capable of binding to m7GTP. While removal of
phosphorylation events does not indicate any phenotypic characteristics. This hints at several
mRNA’s and thereby proteins that are up/down-regulated in mutant eIF4E so far not studied or
reported. We aim at finding out these mRNA’s and proteins using deep sequencing and
SILAC, responsible for giving characteristic phenotypic properties. This study will focus on
significance of eIF4E activity during progression from budding to filamentous growth of yeasts
and will help to design strategies against invasive growth of fungal infections.
Page 236
Poster Abstracts
201
nELAVL mediated RNA regulation during Alzheimer's Disease
Claudia Scheckel1, Elodie Drapeau2, Maria Frias1, Joseph Buxbaum2, Robert Darnell1
1 Rockefeller University, United States of America
2 Mount Sinai School of Medicine, United States of America
Presenter: Claudia Scheckel
Neuron-specific ELAV-like (nELAVL) RNA binding proteins have been linked to numerous
neurodegenerative disorders, but the targets of nELAVL in the human nervous system are still
largely unknown. Using HITS-CLIP we globally identified nELAVL RNA binding sites in healthy
and diseased human brain. We found that, similarly to the mouse homolog, human nELAVL
binds U-rich sequences located within 3’UTRs and introns. Validating these findings, many
robust nELAVL binding sites are conserved between humans and mice, and GO term analysis
of nELAVL targets revealed that transcripts with nELAVL binding sites are most enriched for
terms related to neuronal function and synaptic transmission. To investigate whether there are
changes in nELAVL-mediated RNA regulation during Alzheimer’s Disease (AD) progression, we
generated nELAVL HITS-CLIP binding maps from brain tissue of patients with early and
advanced AD. We observed differential nELAVL binding between healthy and diseased
patients, both within 3’UTRs and introns of transcripts that are linked to neurological function.
The most significant change of nELAVL binding in AD patient samples was a dramatic increase
in its association with Y RNAs, a class of non-coding RNAs. Interestingly, the composition of Y
RNPs is known to be modulated by stress. Because the abundance of Y RNAs is largely
unchanged between healthy and AD patients, our results suggest that Y RNA complexes are
remodeled during AD progression, and this process may be regulated by the nELAVL proteins.
Page 237
EMBO Conference Series: Protein Synthesis and Translational Control
202
Proteome-wide analysis of the nascent chain interactome of the signal
recognition particle by selective ribosome profiling
Daniela Schibich, Günter Kramer, Bernd Bukau
DKFZ-ZMBH Alliance, Germany
Presenter: Daniela Schibich
Many factors contact nascent polypeptide chains when they emerge from the ribosomal exit
tunnel. These interactions determine the fate of the newly made proteins. In E. coli , nascent
polypeptides destined for translocation into or across the membrane are targeted to the
translocation pore SecYEG by two different pathways facilitating either co- or posttranslational
translocation. Co-translational protein translocation directly links protein synthesis to
translocation. It is initiated by binding of the signal recognition particle (SRP) to translating
ribosomes at very early stages during translation before the nascent chain emerges from the
ribosomal tunnel. This SRP interaction is highly transient and stabilized only by the presence of
a hydrophobic signal (anchor) sequence in the nascent chain. Upon interaction with the SRP
receptor (FtsY), SRP targets the translating ribosome to the translocon. This docking aligns the
ribosomal exit tunnel with the conducting channel of the SecYEG complex, allowing the
substrate to pass through or to integrate into the cytoplasmic membrane. In the
post-translational pathway, proteins are translocated by the SecA/SecB system. In this study
we identified the SRP interactome and studied the coordination of SRP-ribosome interaction
with the translation process using “selective ribosome profiling”. The method is based on
purification of SRP bound to ribosome-nascent chain complexes and subsequent identification
of the length and identity of the nascent polypeptide by deep sequencing of the mRNA
fragments protected from nuclease treatment by the translating ribosome. We show that SRP
is almost exclusively recruited to nascent integral membrane proteins and will provide
proteome-wide data demonstrating how ribosome recruitment of SRP is controlled by the
localization of hydrophobic signal anchor sequences in the primary structure of nascent
polypeptides. Our study provides the first comprehensive identification of the SRP interactome.
Page 238
Poster Abstracts
203
IRES-dependent translation of cyp24A1 is controlled by PI3K-Akt signaling
Daniela Rübsamen, Michael Kunze, Victoria Buderus, Thilo Brauß, Magdalena Bajer,
Bernhard Brüne, Tobias Schmid
Goethe-University Frankfurt, Germany
Presenter: Tobias Schmid
Inflammatory conditions have been shown to contribute to tumor progression. Yet, while the
influence of an inflammatory tumor microenvironment on transcriptional changes in tumor cells
is widely studied, translational changes remain largely elusive. In the current project, we aimed
at identifying novel translationally regulated targets during inflammation-associated
tumorigenesis. To this end we co-cultured MCF7 breast tumor cells with conditioned medium
of activated macrophages (CM) and determined translational changes via polysomal
fractionation and microarray analysis. Here, we show that the translation of cytochrome P450
24a1 (cyp24a1) increased in response to inflammatory conditions. We further determined the
presence of an IRES element within the 5'UTR of cyp24a1 mRNA. Cyp24a1 IRES activity was
induced by CM in a PI3K-dependent manner and inhibition of PI3K attenuated polysomal
association of cyp24a1 mRNA. Moreover, overactivation of Akt sufficed to enhance cyp24a1
IRES activation. So far, cyp24a1 has been considered to be primarily regulated transcriptionally
via the vitamin D receptor. At the same time, cyp24a1 inactivates active vitamin D, thereby
establishing a negative feedback loop. As a consequence, clinical applications of active vitamin
D for breast cancer treatment are hampered due to the development of resistances. The
identification and characterization of the PI3K-Akt-responsive IRES-dependent translation as a
new mode of cyp24a1 regulation, might open novel routes to overcome vitamin D resistances
and, thus, to retain therapeutic efficacy.
Page 239
EMBO Conference Series: Protein Synthesis and Translational Control
204
A G-quadruplex within the 5’ UTR of the acid-sensitive potassium leak
channel, TASK-3, determines mRNA fate and membrane expression of the
channel.
James Schofield, Joanne Cowan, Ita O'Kelly, Mark Coldwell
University of Southampton, United Kingdom
Presenter: James Schofield
Regulated expression of the acid-sensitive potassium leak channel, TASK-3, is essential for
normal neuronal network behaviour. TASK-3 is highly expressed in neuronal tissues and a
mutated TASK-3 gene results in Birk Barel mental retardation dysmorphism syndrome. We
have found that post transcriptional regulation is determined by a guanine-rich 42 nucleotide
sequence, predicted to form a stable G-quadruplex, at the 5’ terminus of the mRNA. Directed
mutation of the G-quadruplex-forming sequence resulted in highly modified translation of
TASK-3 protein in HeLa cells. G-quadruplex structures have been shown to be targets of
RNA-binding proteins, affecting mRNA subcellular targeting and promotion or inhibition of
translation initiation. We are investigating potential roles of important neurological RNA-binding
proteins with G-quadruplex-specific activities. The G-quadruplex within the 5’UTR of TASK-3
mRNA predicts specific post-transcriptional regulation of cell-surface TASK-3 channel
expression. Dysregulation of this system is likely to significantly alter neurophysiology.
Page 240
Poster Abstracts
205
Translational control of specific mRNAs is important for cellular survival and the
anti-inflammatory feedback during macrophage activation
Johanna Schott1, Janine Philipp1, Heiner Schäfer2, Georg Stoecklin1
1 German Cancer Research Center, Germany
2 Christian-Albrechts-University of Kiel, Germany
Presenter: Johanna Schott
We analyzed mRNA translation during the course of macrophage activation by recording
polysome profiles from RAW264.7 macrophages that were stimulated with lipopolysaccharide
(LPS). At the global level, we found that the rate of total protein synthesis increases in the early
phase of activation, followed by a decrease in the late phase. Inhibition of translation in the late
phase correlates with phosphorylation of eukaryotic translation initiation factor 2, a central
regulator of translation initiation. To identify individual mRNAs whose translation is specifically
regulated during macrophage activation, mRNAs from different parts of the polysome profile
were quantified by microarray analysis. After one hour of LPS treatment, the strong activation
at the level of translation was observed for four inhibitors of the NFkB signaling pathway,
Nfkbid (IkB-delta), Nfkbiz (IkB-zeta), Ier3 (IEX-1) and Nr4a1, a transcription factor for IkB-alpha.
Posttranscriptional inhibitors of cytokine expression such as Zc3h12a (Regnase-1) and Zfp36
(TTP) were also translationally activated, as well as the mRNA encoding the potent
pro-inflammatory cytokine TNF. In resting cells, these mRNAs had a significantly lower
polysome association than the average mRNA with a similar open reading frame length,
suggesting that their translation is repressed prior to stimulation. In addition, transcriptome
analysis by RNA-Seq revealed a very high correlation between the expression profiles of Ier3
and TNF during macrophage activation. The analysis of BMDM from Ier3 knockout mice
showed that Ier3 protects macrophages from LPS-induced cell death, which was previously
described to result from auto-/paracrine secretion of TNF. Taken together, our analysis reveals
that feedback inhibitors are co-regulated with pro-inflammatory genes and that translational
control during macrophage activation is important for cellular survival and the resolution of
inflammation.
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EMBO Conference Series: Protein Synthesis and Translational Control
206
Translational control of proto-oncogene expression by upstream open reading
frames (uORFs)
Julia Schulz, Klaus Wethmar, Enrique M. Muro, Nancy Mah, Miguel A. Andrade-Navarro,
Achim Leutz
Max-Delbrueck-Center for Molecular Medicine, Germany
Presenter: Julia Schulz
Quantitative induction and functional activation of proto-oncogenes are associated with human
cancer, yet the reason for increased proto-oncogene expression often remains obscure. Here
we characterized the impact of upstream open reading frame (uORF)-mediated translational
control on the expression of tyrosine kinases (TKs) and other selected proto-oncogenes.
Sequence analyses identified one or several uORF(s) in 89 out of 140 human TKs (64%). TK
uORFs tended to be conserved among human and mouse. Functional deletion of uORF
initiation codons by introducing an AUG to UUG point mutation in ten TKs and four
proto-oncogenes unanimously resulted in enhanced downstream translation of reporter
constructs. Moreover, experimental deletion of uORF-related stop codons caused a
pronounced inhibition of downstream translation if the elongated uORF overlapped the main
coding sequence (CDS). Thus, translation of many proto-oncogenes is repressed through
uORFs. Naturally occurring loss-of-uORF alleles of MET and KDR were also found to be
associated with increased translation of the CDS in vitro. Bioinformatics uncovered
polymorphic uORF start codons and polymorphic uORF-related Kozak consensus sequences
in 5,7% and 14,3% of TK genes, respectively. These results imply that loss-of-uORF mutations
may represent a widespread, yet currently unappreciated mechanism of oncoprotein
activation. To experimentally search for cancer-related uORF mutations, we have established a
two-step, multiplex identifier (MID)-tagged PCR approach, allowing simultaneous
re-sequencing of 400 individual uORFs amplified from 300 human tumors. We anticipate that
our systematic screen will resolve whether or not uORF deletions by genetic polymorphisms or
de-novo mutations increase tumor susceptibility and promote malignant transformation in
humans.
Page 242
Poster Abstracts
207
RNP capture of defined RNA species in vivo
Birgit Schuster, Alfredo Castello, Matthias Hentze
EMBL Heidelberg, Germany
Presenter: Birgit Schuster
In response to many stimuli, changes in biological conditions or stress, the translation of RNA
into proteins is regulated by RNA binding proteins (RBPs). The identification and
characterization of RBPs bound to defined RNA species greatly impacts on our understanding
of translational control mechanisms. Recently, a comprehensive and unbiased method, called
mRNA interactome capture, was developed for the global identification of RBPs bound to
polyadenylated RNAs in living cells [1; 2; 3]. The design of this method focuses on the
determination of complete mRNA interactomes, but cannot be applied to capture RBPs
assembled on defined RNA species. While methods to identify RNAs bound by single RBPs
are available (e.g. CLIP, PAR-CLIP, HITS-ClIP [reviewed in 4] identifying RBPs that bind a
particular RNA has been challenging so far. Here we present an experimental approach to
determine the RBPs bound to a defined RNA species in vivo. Using the global mRNA
interactome capture approach including in vivo UV crosslinking as a starting point, we tailor the
pull-down protocol towards specific RNAs by making use of DNA/LNA mixmer
oligonucleotides bound to magnetic beads. Our proof of principle model is based on luciferase
reporter constructs that allow introduction of known RNA motifs upstream or downstream of
the targeted open reading frame. We will discuss performance parameters of our approach to
meet an urgent need in RNA research.
References:
[1] Castello A, et al. Cell. 2012; 149 (6) [2] Baltz AG, et al. Mol. Cell 2012 46 (5) [3] Castello A,
et al. Nature Protoc. 2013; 8 (3) [4] Milek M, et al. Cell Dev.Biol. (2012) 23 (2)
Page 243
EMBO Conference Series: Protein Synthesis and Translational Control
208
A systems analysis of translation in totipotent stem cells
Juliane Schwarz, Sebastian Leidel
MPI for Molecular Biomedicine, Germany
Presenter: Juliane Schwarz
Stem cells are pluripotent and can differentiate into different cell types. Embryonic stem cells
(ESC) exhibit the greatest potential of differentiation and can develop into cell-types of all three
germ layers. These characteristics made them very attractive for regenerative medicine but
have also raised ethical concerns. Induced pluripotent stem (iPSC) may have solved this
dilemma. iPSC are derived from adult somatic cells, which are reprogrammed into a totipotent
state by the forced expression of reprogramming factors. iPSC are similar to ESC in many
aspects. However, they also exhibit clear differences in gene expression and may retain a
partial epigenetic memory characteristic of the somatic cell of origin. The extent of these
differences is not fully understood and we know little about the translational programs of ESC
and iPSC. However, it is crucial to understand this program in iPSC in detail to use these cells
as a tool to model translational diseases. To shed light into these questions, I apply ribosome
profiling in murine and human ESC and iPSC. First, using polysome profiling I established the
conditions for optimal lysis and digestion with RNaseI in all four cell lines. Second, combining
inhibitors, run off experiments and Western Blot I verified these conditions. Finally, I compared
different digestion protocols by sequencing libraries of ribosome protected fragments (RPF).
Comparing read numbers, RPF length and the extent of rRNA contamination I found that a
slight overdigest is the optimal treatment of the samples. Finally, I analyzed the translational
state of iPSC and ESC using the RPF sequencing data. Interestingly, I found differentially
translated targets, which I am currently analyzing in detail. Our work will further our
understanding of translational programs in stem cells. Importantly, this will allow us to better
model translational diseases in the future.
Page 244
Poster Abstracts
209
Ribosomal protein hydroxylation is a new post-translational modification of
translational machinery conserved from prokaryotes to humans
Rok Sekirnik1, Wei Ge1, Alex Wolf2, Adam Zayer1, Mathew Coleman1, Christopher J.
Schofield1
1 University of Oxford, United Kingdom
2 Institute of Molecular Toxicology and Pharmacology, HelmholtzZentrum Munchen,
Germany
Presenter: Rok Sekirnik
Post-translational hydroxylation of ribosomal proteins has recently been identified as a new
oxygen-dependent modification of translational machinery. The 2-oxoglutarate
(2OG)-dependent oxygenase ycfD from Escherichia coli catalyzes post-translational
hydroxylation of Arg81 on the 50S ribosomal protein L16, which is located in the immediate
proximity of the peptidyl transferase centre (PTC). We demonstrated that ycfD affects growth
and global translation rates, as well as susceptibility to ribosomal antibiotics. YcfD-catalyzed
ribosomal protein hydroxylation in prokaryotes is regulated by availability of oxygen and
nutrients, thus presenting a potential interface between metabolism and translation. Mass
spectrometric, structural and evolutionary studies provide evidence that ycfD is the bacterial
progenitor of human ribosomal protein hydroxylases NO66 and MINA53, which are linked to
cancer progression. Mechanistic understanding of ribosomal protein hydroxylation also
provides a new paradigm for the design of selective inhibitors of modified ribosomes, found in
tissues with limited nutrient availability, such as cancer.
Page 245
EMBO Conference Series: Protein Synthesis and Translational Control
210
Designing a stress resistant translation machinery - lessons from
trypanosomatids
Alexandra Zinoviev1, Melissa Leger2, Gerhard Wagner2, Michal Shapira1
1 Ben-Gurion University of the Negev, Israel
2 Harvard Medical School, United States of America
Presenter: Michal Shapira
Extreme temperatures cause a global translation arrest, preventing the accumulation of
polypeptides that risk misfolding. This is common in digenetic parasites that migrate between
invertebrates and mammals. Since temperature and pH switches drive the developmental
program of gene expression in trypanosomatids, both canonical and alternative pathways for
translation are required. Under conditions that mimic transmission to the host, the canonical
translation initiation complex (LIF4E-4) disintegrates, and an alternative cap-binding complex
comes into play (LIF4E-1), pulling down a multitude of initiation factors, except for any eIF4G
ortholog. We have obtained crystals of LIF4E-1, in attempt to solve its unusual mode of
regulation. Trypanosomatids do not express any homolog of 4E-BP, but a novel and
non-conserved 4E-Interacting protein (L4E-IP, 85 kDa) regulates the binding of LIF4E-1. A
nutritional stress experienced within the fly is known to induce parasite virulence, and prepare
for stage differentiation. Another paralog, LIF4E-3, has been assigned a function during
starvation. A mutation in the cap-binding pocket impairs its cap-binding activity, preventing its
ability to compete with the canonical factors. Instead, it enters into granules that are induced
during starvation, most probably to accompany and protect inactive RNAs. LIF4E-3 is
regulated by a novel eIF4G-like protein that under normal conditions sequesters it in the
cytoplasm, and releases it during nutritional stress to enter specific stress granules that are
devoid of DHH1. We highlight how orthologs of conserved factors have been recruited during
evolution to acquire novel functions that promote survival under harsh conditions.
Page 246
Poster Abstracts
211
Validation of a cell-based high-throughput screening assay for
posttranscriptional regulation
Valentina Adami, Alessandro Quattrone, Viktoryia Sidarovich
CIBIO, University of Trento, Italy
Presenter: Viktoryia Sidarovich
Posttranscriptional regulation, specifically exerted through a number of cis-acting elements
residing mainly in mRNA 3' untranslated regions (3'UTRs), has a profound impact on gene’s
expression. However, screening assays commonly focus on transcriptional regulation, i.e. the
identification of promoter-targeting molecules. Here we describe the development and
validation of a cell-based assay to investigate the role of a 3'UTR in the modulation of its
mRNA fate, and to identify compounds able to affect it. MYCN amplification in neuroblastoma
patients is strongly associated with advanced disease stages, rapid tumour progression and
poor prognosis, making this gene an obvious therapeutic target. Both computational studies
and experimental evidence point out high level of conservation in the 3'UTR sequence
enriched in potentially functionally cis-acting elements, thus indicating for a highly regulated
sequence. The luciferase reporter constructs with full length MYCN 3'UTR were generated and
subsequently stably integrated in a neuroblastoma cell line. A screening was carried out using
a 2000 compound library including about 1000 of the FDA-approved drugs, with luciferase
activity assessed after 24 hours of a 2 μM treatment. The identified hits were checked for
reproducibility and counterscreened for promoter effects and cytotoxic activity. The
counterscreening selected 4 compounds as truly dependent on MYCN 3'UTR, one of which
was further validated through low-throughput technologies. We suggest this reporter gene
assay as a valuable tool to screen chemical libraries for compounds modulating
posttranscriptional control mechanisms.
Page 247
EMBO Conference Series: Protein Synthesis and Translational Control
212
Comprehensive profiling of the eIF2a-mediated unfolded protein response
Carmela Sidrauski1, Anna McGeachy2, Nicholas Ingolia3, Peter Walter1
1 UCSF, United States of America
2 Johns Hopkins, United States of America
3 Carnegie Institution for Science, United States of America
Presenter: Anna McGeachy
Translational control of gene expression provides quick, reversible responses to many cellular
stresses. Several different stress signals converge on the phosphorylation of the translation
initiation factor eIF2α. This phosphorylation event, which is conserved from yeast to humans,
decreases global protein synthesis while paradoxically inducing the expression of specific
target genes. It is required for normal cellular physiology, for example in pancreatic islet beta
cells, but its pathological activation has also been implicated in diseases such as
neurodegeneration. We characterized the full landscape of eIF2α-mediated translational
changes in the unfolded protein response (UPR), which is triggered by defects in the folding of
secretory proteins, using ribosome profiling and RNA-Seq. Our data reveal novel UPR target
transcripts, including mRNAs whose function seems specific to secretory protein mis-folding
despite the role of eIF2alpha phosphorylation in many other stresses. The translational UPR
that we observe depends wholly on eIF2α phosphorylation; deletion of the ER-resident eIF2α
kinase PERK abolishes both the translational and the rapid transcriptional components of the
response. Our data also suggest a role for eIF2α phosphorylation in coordinating the UPR
gene expression program. Non-phosphorylatable eIF2α does not disrupt the transcriptional
response but does reduce the translation of induced mRNAs. These results suggest an
additional role for stress-inducible translational control.
Page 248
Poster Abstracts
213
Characterization of the eIF4E-binding protein, 4E-T, in mammalian cells
Clare Simpson, Nancy Standart
The University of Cambridge, United Kingdom
Presenter: Clare Simpson
For efficient translation to occur the 5’cap bound eIF4E forms a ‘closed loop complex’ with the
poly (A) tail, via PABP binding to eIF4G. 4E-T(ransporter) is an example of an eIF4E-binding
protein which competitively inhibits eIF4G binding to eIF4E by binding to the same domain,
thus preventing translation. Work in our laboratory has also shown that surprisingly, 4E-T
represses translation of tethered mRNA in an eIF4E-independent manner [1]. Cup, the
Drosophilia melanogaster homologue of 4E-T, is crucial for its germ line development via its
regulation of specific mRNAs including nanos and oskar [2]. However, despite the importance
of Cup, little equivalent information is known about 4E-T, including which other proteins it
binds to and most importantly which mRNAs it may be selectively targeting. To try to address
some of these questions we have taken a multifaceted approach to studying the role of 4E-T in
mammalian cells. The first, is by changing the levels of 4E-T to ask what differences occur in
the mRNAs being translated using polysomal profiling and sequencing analysis. The second is
by using mass spectrometry to identify proteins bound to 4E-T after immunoprecipitation.
References:
[1] Kamenska A., et al., unpublished work [2] Piccioni F. et al., (2005) RNA Biology.
2(4):125-128
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EMBO Conference Series: Protein Synthesis and Translational Control
214
The mechanism of translation initiation of the unspliced HIV-1 mRNA
Victoria Smirnova, Ilya Terenin, Anastasia A. Khutornenko, Dmitry Andreev, Sergey
Dmitriev, Ivan Shatsky
Lomonosov Moscow State University, Russian Federation
Presenter: Victoria Smirnova
The authentic translation initiation mechanism of unspliced HIV-1 mRNA remains disputable.
While this mRNA is naturally capped and there is little doubt that it can be translated via the
cap-dependent mechanism, numerous reports stated that it also may initiate translation in a
cap-independent fashion. The first paper to announce existence of an Internal Ribosome Entry
Site in the leader of HIV-1 unspliced mRNA was published about a decade ago. Since then
this issue has remained controversial, being confirmed in some reports but not validated in
others. The consensus of opinions is that HIV-1 5’UTR IRES functions when cap-dependent
translation is inhibited; more specifically it was suggested to be active in G2/M-phase.
However, the question of which mechanism prevails, if any, is still unclear. Here we have
addressed the potential of the unspliced HIV-1 mRNA 5‘UTR to function as an IRES using our
previously reported stringent criteria, which ground on direct comparison of monocistronic and
bicistronic mRNAs translation efficiency. Such an approach enables one to address
contribution of both mechanisms to the overall level of capped monocistronic mRNA
translation. Importantly, we have committed to transfections of RNA, rather then DNA, given
the latter approach’s strong bias towards producing artifacts. We were able to show that
under all conditions tested the primary mechanism used by 5’-UTR of HIV-1 unspliced mRNA
is 5’-end dependent scanning. Absence of an IRES and relatively high cap-dependence of
RNA with this leader were confirmed both in vitro (translation in cell lysates) and in vivo
(RNA-transfection of several cell lines, including those derived from T-cells). Hypothesis of cell
cycle dependent IRES, which is active in G2/M phase when cap-dependent translation is
suppressed, has also been disproved with transfection of synchronized cells or under
conditions when cap-dependent translation was directly inhibited.
Page 250
Poster Abstracts
215
Investigation of the translational regulation of terminal oligo pyrimidine tract
(TOP) containing mRNAs
Amandine Bastide, Samantha Johnston, Martin Bushell, Anne Willis, Ewan Smith
MRC, Toxicology Unit, United Kingdom
Presenter: Ewan Smith
Ribosomal biogenesis is coupled to nutrient availability, cell growth and differentiation status.
The mRNAs encoding ribosomal proteins and the cognate translation factors involved in
protein synthesis are regulated specifically in tune with these cellular conditions. Translational
regulation of these transcripts is conferred by a cis-regulatory element at the beginning (5’) of
the mRNAs, known as the terminal oligo pyrimidine (TOP) tract. This motif consists of a
C-residue modified with m7-guanosine triphosphate (cap) followed by 4-14 pyrimidine bases.
The presence of the pyrimidine tract confers a specific control mechanism for this subset of
mRNAs, allowing for coordinated repression of ribosomal biogenesis at times when cells
require energy conservation and thus growth inhibition. Despite many attempts to elucidate
them, the mechanisms by which these messages are regulated and the trans-acting factors
involved in this specific regulation have remained elusive. Here we utilise luciferase reporter
constructs containing TOP tracts in their 5’UTR, combined with RNA affinity purification
techniques and mass spectrometry to develop a system to identify TOP tract binding proteins.
By combining these methods with mutations in the oligo pyrimidine tract it will be possible to
identify factors that alter their binding when nutrients are limited or when mutations prevent
interaction. Presented here are our initial purification attempts and insights into how to develop
a sensitive and specific method to purify TOP binding proteins.
Page 251
EMBO Conference Series: Protein Synthesis and Translational Control
216
Structural basis for species-specific polypeptide-mediated translational arrest
Daniel Sohmen1, Michael Habeck2, Charlotte Ungewickell1, Otto Berninghausen1, Roland
Beckmann3, Daniel N. Wilson4
1 Gene Center and Department for Biochemistry, Germany
2 MPI for Developmental Biology - Tübingen, Germany
3 Gene Center and Department for Biochemistry, and Center for integrated Protein Science
Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodo, Germany
4 Gene Center Munich, Germany
Presenter: Daniel Sohmen
Ribosomal Ribosomal protein synthesis occurs through the stepwise addition of amino acids
to a growing polypeptide chain in a linear fashion. The chain of amino acids grows from the
peptidyl transferase center (located on the large subunit of the ribosome), passes through a
90-100Å wide tunnel and exits to the solvent side. Some polypeptides such as the Bacillus
subtilis MifM leader peptide can induce stalling of the ribosome during translation, thereby
allowing translation of the downstream open reading frame yidC2 [1], and thus providing an
additional level of gene regulation. The mifM open reading frame encodes a small protein of 94
amino acids with a stalling sequence causing translational arrest of the ribosome by interacting
with components of the ribosomal tunnel [2]. Most interestingly MifM arrests translation on
Bacillus subtilis ribosomes but not on Escherichia coli ribosomes [3,4], despite the high degree
of conservation of the ribosome and the translation machinery. Here we present a structure of
the MifM-stalled Bacillus subtilis ribosome at a resolution approaching 4Å, determined by cryoelectron microscopy and single-particle reconstruction. The structure provides structural
insight into the species-specific communication pathways existing between the nascent
polypeptide chain and the ribosomal tunnel in B. subtilis compared to E. coli.
References:
[1] Chiba, S., Lamsa, A., & Pogliano, K. (2009). A ribosome-nascent chain sensor of
membrane protein biogenesis in Bacillus subtilis. EMBO; [2] Wilson, D. N., & Beckmann, R.
(2011). The ribosomal tunnel as a functional environment for nascent polypeptide folding and
translational stalling. Current opinion in structural biology; [3] Chiba, S., Kanamori, T., Ueda, T.,
Akiyama, Y., Pogliano, K., & Ito, K. (2011). From the Cover: Recruitment of a species-specific
translational arrest module to monitor different cellular processes. PNAS; [4] Vázquez-Laslop,
N., & Mankin, A. S. (2011). Picky nascent peptides do not talk to foreign ribosomes. PNAS
Page 252
Poster Abstracts
217
The influence of stop codon 3'-context on competition between termination
factors and supressor tRNAs
Elizaveta Sokolova1, Boris Eliseev1, Peter Vlasov2, Elena Alkalaeva1
1 Engelhardt Institute of Molecular Biology Russian Academy of Sciences, Russian
Federation
3 Centre for Genomic Regulation and Universitat Pompeu Fabra, Barcelona, Russian
Federation
Presenter: Elizaveta Sokolova
Today there is a set of evidences about influence of the nearest stop codon environment on
translation termination efficiency both in prokaryotes and eukaryotes. Earlier in our laboratory it
was shown that 3’-contexts of stop codons from tobacco mosaic virus (TMV) and human
dystrophine gene in different ways affected readthrough in cells, containing chimeric eRF1s (B.
Eliseev et al. 2011). In this work we studied how these contexts in combination with different
stop codons influenced efficiency of translation termination in completely reconstructed in vitro
eukaryotic translation system. As a control we used standard 3’-context calculated using
bioinformatics analysis of nucleotide frequences in human genome. After addition of supressor
tRNAs at the same time with termination factors to the system it became clear that three stop
codons, followed with TMV as well as standard context, were not equally susceptible to
supressor tRNA recognition.On TMV context the contribution of UAA and UGA stop codons to
translation termination efficiency changed in comparison with a standard context. We observed
greater difference between these two stop codons on TMV context, than on the standard.
Page 253
EMBO Conference Series: Protein Synthesis and Translational Control
218
4E-T represses translation of tethered mRNAs in a P-body- and eIF4Eindependent manner, and enhances silencing of microRNA-target mRNAs
Anastasiia Kamenska1, Wei-ting Lu2, Nicola Minshall1, Dorota Kubacka1, Martin Bushell2,
Nancy Standart1
1 University of Cambridge, United Kingdom
2 MRC Toxicology Unit, United Kingdom
Presenter: Anastasiia Kamenska
Human 4E-T(ransporter), a relatively poorly characterised nucleocytoplasmic shuttling
eIF4E-binding potein, is enriched in P-(rocessing) bodies, cytoplasmic foci which contain
mRNA, microRNA, RNA degradation machinery and RNA-binding proteins that repress
translation. eIF4E is the only translation initiation factor in P-bodies, and 4E-T the only
eIF4E-binding protein. To understand the function of 4E-T in gene expression control in
mammalian tissue culture cells, we first delineated a conserved C-terminal region as being
responsible for its P-body localisation. We verified in the yeast two hybrid system, by GFP-Trap
immunoprecipitation and by immunofluorescence that eIF4E interacts with the Y 30 TKEELL
motif of 4E-T. Wild type 4E-T, but neither Y 30 A 4E-T nor 4E-TΔC recruits endogenous eIF4E
to P-bodies. Using 35 SMet incorporation, we showed that wild type but not Y 30 A 4E-T
reduced global protein synthesis. In the λN-BoxB tether function assay, 4E-T reduces bound
Renilla luciferase mRNA activity (relative to control Firefly luciferase mRNA) approx. 3-4x, at the
level of translation, and not mRNA decay, as determined by qPCR, irrespective of P-body
localisation. Surprisingly, a similar reduction in expression is seen when Y 30 A or Y 30 LL-AAA
4E-T is tethered to reporter mRNA. Moreover, the N-terminal portion (1-180 aa) of 4E-T which
binds eIF4E (and which as predicted reduces both luciferase activities) is not sufficient to
promote additional repression of tethered mRNA. Last, using RNAi, 4E-T depletion resulted in
~ 55% increased rates of global protein synthesis, and in the relief of microRNA-mediated
repression. Altogether we conclude that 4E-T inhibits global protein synthesis by sequestering
eIF4E, represses translation of bound mRNAs in an eIF4E-independent manner (as also
recently shown for Drosophila Cup [1], and enhances silencing of reporter and cellular
microRNA-target mRNAs.
References:
[1] Igreja C and Izaurralde E (2011). Genes Dev. 25:1955-67
Page 254
Poster Abstracts
219
eIF1 phosphorylation mediates leaky scanning translation initiation
Lolita Tzach, Ariel Stanhill
Technion, Israel
Presenter: Ariel Stanhill
Accurate protein synthesis is required in order to establish a correct proteome. Initial steps in
protein synthesis are highly regulated processes as they define the reading frame of translation
and commit the translation machinery to begin the elongation phase. Eukaryotic translation
initiation is a process facilitated by numerous factors (eIFs), aimed to form a “scanning”
mechanism to the initiation codon. Translation initiation has been reported to be regulated by
re-initiation and leaky scanning. Re-initiation regulation is governed by the phosphorylation
status of eIF2alpha and controlled by cellular stresses via the Integrated Stress Response (ISR)
pathway. However, regulation of the leaky scanning translation initiation is not known to be
regulated or connected to cellular conditions. We have identified AIRAP to be translationally
induced in a leaky scanning regulatory manner. AIRAP transcript contains a single upstream
Open Reading Frame (uORF) in a poor-kozak context. Translation induction during stress
conditions is governed by means of leaky scanning and not re-initiation. This induction of
AIRAP is solely dependent on eIF1 and the uORF kozak context. We show that eIF1 is
phosphorylated under specific conditions that induce protein misfolding and have
biochemically characterized this site of phosphorylation. Our data indicate that leaky scanning
like re-initiation is responsive to stress conditions and that leaky scanning can govern ORF
translation induction by bypassing poor kozak context of a single uORF transcript.
Page 255
EMBO Conference Series: Protein Synthesis and Translational Control
220
ABC50 plays a critical role in start-site selection during mRNA translation
Joanna Stewart, Joanne Cowan, Mark Coldwell, Christopher Proud
University of Southampton, United Kingdom
Presenter: Joanna Stewart
The ATP-binding cassette protein, ABC50 (also termed ABCF1), was identified as interacting
with eukaryotic initiation factor eIF2, the protein which recruits the initiator methionyl-tRNA to
the small (40S) ribosomal subunit. It is the anticodon of this tRNA which recognises the start
codon of the mRNA. Previous data have indicated that ABC50 plays a positive role in the
initiation of both general, 5’-cap-dependent translation and translation driven by certain internal
ribosome entry sequences. However, the nature of the role of ABC50 in translation initiation
has remained obscure. Expression of mutants of ABC50 which are defective for ATP binding
or hydrolysis, or siRNA-mediated knock down of ABC50, in HEK293 cells has allowed more
detailed investigations into the role of ABC50 in translation initiation. We show that defective
ABC50 decreases overall translation and also results in an altered pattern of start codon
selection. Furthermore, analysis of the interplay between ABC50 with eIF2 and other key
initiation factors involved in start-site selection provides new insights into the function and
importance of ABC50 in translation initiation. This work is supported by funding from BBSRC.
Page 256
Poster Abstracts
221
A proteomic analysis of changes in the RNA binding protein interactome during
chemotherapeutic stress
Mark Stoneley, Rebekah Jukes-Jones, Kelvin Cain, Martin Bushell, Anne Willis
Medical Research Council, United Kingdom
Presenter: Mark Stoneley
Chemotherapeutic agents provoke multiple stress response pathways that ultimately lead to
cell death or senescence. It is clear that post-transcriptional control of gene expression plays a
major role in the cellular response to stress. Signalling through stress response pathways can
impact on many post-transcriptional mechanisms, including alternative splicing, mRNA decay
and mRNA translation. To gain a more comprehensive understanding of the
post-transcriptional response to chemotherapeutic stress, we are currently identifying
stress-mediated changes in the RNA binding protein interactome using in vivo crosslinking
coupled with RNA binding protein capture. In untransformed epithelial cells, we find that the
chemotherapeutic agents, doxorubicin and actinomycin D, inhibit ribosomal RNA transcription
resulting in activation of the nucleolar stress response pathway. In addition doxorubicin
stimulates rapid and widespread genomic DNA damage. Thus we are using these agents to
gain insight into the post-transcriptional response to nucleolar stress and DNA strand breaks.
Given the crucial role that RNA-protein interactions play in post-transcriptional control, we are
currently performing a quantitative proteome-wide analysis of the changes in RNA-protein
association during actinomycin D and doxorubicin toxicity. We have successfully employed in
vivo UV crosslinking and oligo(dT) affinity capture to isolate RNA binding proteins. Purified RNA
binding proteins are subsequently subjected to quantitative tandem mass spectrometry
(LC-MS/MS) to identify proteins that display altered RNA binding characteristics during cell
stress. This approach will undoubtedly reveal novel pathways for post-transcriptional control of
gene expression during cell stress and lead to an enhanced understanding of the role of RNA
binding proteins in the cellular response to DNA damage and nucleolar stress.
Page 257
EMBO Conference Series: Protein Synthesis and Translational Control
222
The phosphoproteome of ribosomes
Katharina Brünger1, Juliane Horenk1, Benno Ehrl1, Igor Paron2, Matthias Mann2, Katja
Sträßer1
1 LMU Munich, Germany
2 MPI of Biochemistry, Germany
Presenter: Katja Sträßer
Eukaryotic protein synthesis is a sophisticated process that requires an extensive biological
machinery and diverse control mechanisms. Phosphorylation, a prominent mechanism to
regulate a wide variety of cellular processes, controls translation mainly at the level of
translation initiation factors. However, surprisingly little is known about the phosphorylation of
the translation machinery itself, the ribosome, and its effect on translation. We present a
comprehensive catalogue of over 300 phosphosites on ribosomal proteins of the eukaryote S.
cerevisiae. In addition, we report changes in the phosphorylation status under different stress
conditions. Interestingly, using phosphorylation-deficient alanine mutants we show that
ribosomal phosphorylation sites are important for different steps of the translation cycle. In the
future, we aim to elucidate the molecular function of phosphorylated sites in all steps of
translation. Second, we aim to demonstrate the physiological relevance of ribosome
phosphorylation. Taken together, we show that ribosomal proteins are heavily phosphorylated
and that some phosphosites function in translation. Thus, phosphorylation of ribosomal
proteins might be a widely used mechanism to regulate translational activity.
Page 258
Poster Abstracts
223
Regulation of translation initiation during stress conditions in Candida albicans
Arunkumar Sundaram, Chris Grant
Uni of Manchester, United Kingdom
Presenter: Arunkumar Sundaram
Oxidative stress has been implicated in various disease states including cancer, hypertension
and diabetes. An oxidative stress occurs when ROS overwhelm the antioxidant defenses of the
cell. Global inhibition of protein synthesis is a common response to stress conditions including
exposure to ROS. This reduction in protein synthesis may prevent continued gene expression
during potentially error-prone conditions as well as allow for the turnover of existing mRNAs
and proteins whilst gene expression is reprogrammed to deal with the stress. We have
previously analysed the regulation of protein synthesis in response to oxidative stress in the
yeast Saccharomyces cerevisiae. Our data show that oxidative stress induced by hydrogen
peroxide causes an inhibition of translation initiation that is dependent on the Gcn2 protein
kinase which phosphorylates the a- subunit of translation initiation factor eIF2. We also showed
that in contrast to hydroperoxides, inhibition of translation initiation in response to cadmium
and diamide depends on both Gcn2 and the eIF4E binding protein Eap1. In this current study,
we have examined oxidant-specific regulation of translation initiation in the human pathogenic
yeast Candida albicans. Our data show that oxidants cause a rapid inhibition of translation
initiation in C. albicans, as judged by polysome analysis. However, there are differences in
translational responses between S. cerevisiae and C. albicans. Our results indicate that H 2 O
2 and cadmium inhibit initiation in a Gcn2-dependent manner mediated via eIF2αp, while
diamide inhibits inhibition in a Gcn2-independent manner. Furthermore, oxidant induced
inhibition of translation initiation does not depend on Caf20, which is the single eIF4E binding
protein present in C. albicans. We will present our data highlighting the differences in
translational controls between these yeasts.
Page 259
EMBO Conference Series: Protein Synthesis and Translational Control
224
Erythromycin enhances the naturally occurring peptidyl-tRNA drop-off from the
translating ribosome
Vyacheslav Kolb, Maxim Svetlov
Institute of Protein Research, Russian Federation
Presenter: Vyacheslav Kolb
Erythromycin binds to the exit tunnel of the bacterial ribosome and induces dissociation of the
peptidyl-tRNA shortly after beginning of translation. It is assumed that the physical blockage of
the tunnel by the drug underlies such a drop-off event. We used a cell-free translation system
programmed with firefly luciferase mRNA to study the dissociation process. Lengths of the
peptide moiety of peptidyl-tRNAs that drop-off from ribosomes during translation were
determined. The length of the nascent peptides synthesized in the presence of erythromycin
varies from four to nine amino acid residues. The drug generates in the cell-free translation
system a reproducible drop-off pattern where dissociation probability depends on the peptide
length. The synthesis of luciferase in the absence of a drug is also accompanied by
dissociation of short peptidyl-tRNAs from translating ribosomes, although such events are
rather rare in comparison with erythromycin-induced ones. Surprisingly, translation in the
absence of erythromycin generates the same set of dropped-off peptidyl-tRNAs as we have
observed in the experiments with the drug. The identity of the two peptide sets indicates that,
to all appearance, erythromycin stimulates the naturally occurring dissociation of peptidyl-tRNA
from the ribosome rather than induces the peptidyl-tRNA drop-off by forming an impassible
barrier for the nascent peptide.
Page 260
Poster Abstracts
225
The p53 translation initiation and the role of potential IRES element in this
process
Agata Swiatkowska, Agnieszka Gorska, Jerzy Ciesiolka
Institute of Bioorganic Chemistry Polish Academy of Sciences, Poland
Presenter: Agata Swiatkowska
The secondary structure of the 5'-terminal region of p53 mRNA containing potential IRES
(Internal Ribosomal Entry Site) has been determined recently in our laboratory. It has been
shown that both translation initiation codons, for p53 and its delta Np53 isoform, are located at
the helix-bulge junctions. Such structural environment raises the question concerning
mechanism of translation initiation process of both proteins p53 and delta Np53. To find the
answer modified antisense oligonucleotides bearing methyl group at 2' position of all ribose
residues were used in vitro and in vivo in normal and stress conditions to generate steric
hindrance or/and to partly unfold the structure of the 5'-terminal region of p53 mRNA. Such
perturbations were expected to change the level of p53 and delta Np53 isoforms and give the
indication which structural elements of IRES play an important role in translation initiation of
both proteins. It turned out that translation initiation was affected by a few modified oligomers
significantly. The results revealed that one of the structural motifs important to initiate p53
synthesis efficiently is the hairpin U180-A218 and its surroundings. It has been earlier
proposed that this region is a potential binding site for Hdm2 protein and that this interaction
promotes p53 synthesis. We suggest that selected antisense oligomers which bind to this
region might compete with some trans-acting translation factors or might disturb the structure
of the hairpin U180-A218 causing a decrease in p53 protein level. Understanding the
translation process of p53 seems to be useful not only in filling the gaps in our current
knowledge but it might help to develop new therapeutic approaches in many human diseases
which result from dysfunction of p53 and its isoforms. * This work was supported by grant
(HOMING PLUS/2012-6/15) from Foundation for Polish Science to AS.
Page 261
EMBO Conference Series: Protein Synthesis and Translational Control
226
tRanslatome: an R package to portray translational controls hidden in
high-throughput assays
Toma Tebaldi1, Erik Dassi1, Galena Kostoska2, Gabriella Viero3, Alessandro Quattrone1
1 CIBIO - University of Trento, Italy
2 DISI - University of Trento, Italy
3 Institute of Biophysics CNR, Italy
Presenter: Toma Tebaldi
High-throughput technologies have led to an explosion of genomic data available for
automated analysis, enabling to sample simultaneously multiple gene expression levels. This
approach requires bioinformatics techniques suitable to integrate raw information coming from
multiple ‘-omics’ data. It has been recently demonstrated that translational control represents a
widespread phenomenon, with pervasive and previously underestimated regulation
capabilities. While detecting changes in mRNA levels and in the polysomal loading of mRNAs is
experimentally feasible, the computational integration of the transcriptional and the translational
cell portraits is still far from being robustly approached. Here we provide a new computational
suite, implemented as an R package, representing a complete platform for the analysis of data
coming from high-throughput assays at two different ‘-omics’ levels: transcriptome and
translatome, using several statistical methods. The package allows the simultaneous
comparison of differentially translated genes and of the corresponding differentially enriched
biological themes, enabling the analysis of post-transcriptional regulatory elements on the
untranslated regions (UTRs) and giving special attention to the graphical representation of
results. The tool is presented with a case study, showing how it can be used with microarray
and sequencing data to detect the effects of translational controls.
Page 262
Poster Abstracts
227
One mRNA, two mechanisms. The case of HCV IRES.
Zanda V. Bochkaeva, Ilya Terenin, Dmitry Andreev, Sergey Dmitriev, Ivan Shatsky
Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russian
Federation
Presenter: Dmitry Andreev
Two principal modes of translation initiation in eukaryotic cells are cap-dependent scanning
mechanism and internal ribosome entry. It is commonly suggested that a single mRNA
predominantly uses only one of these mechanisms. Here we desсribe the case of an mRNA
that can be translated by the both, and simple changes of the mRNA composition affects their
contribution into the overall translation efficiency of the mRNA. Mechanism of Hepatitis C
mRNA translation initiation is one of the most thoroughly studied examples of Internal
Ribosome Entry Sites (IRESes). HCV IRES covers nucleotides 42-342 of the virus mRNA, but
how sequences laying outside this region influence IRES activity remains a controversial issue.
Here, using reporter monocistronic mRNAs we show that deletion of nts. 1-42 stimulates
translation 2-4 times both in vitro and in vivo. We found that the 5'-truncated HCV IRES (nts.
42-342) has the same affinity to the 40S ribosomal subunit or eIF3 as the wild-type IRES.
Interestingly, translation directed by the complete HCV 5' UTR can be stimulated by m 7
G-capping, while that of the 5'-truncated IRES variant can't be. We found that the 5'-cap
stimulates translation of any HCV mRNA variant if it only has any native or artificial sequence
upstream of domain II of the HCV IRES. In order to explain the fact of different
cap-dependence we discriminate between the two modes of translation initiation by
introducing an additional in-frame AUG codon into the HCV coding region downstream from
the authentic one, and by creating the mutated IRES variants. In accordance with our
expectations, the IRES directs translation from the authentic AUG only, while the 5'-end
initiation mode contributes to the translation from both AUGs, irrespectively of the presence of
the 5'-cap.
Page 263
EMBO Conference Series: Protein Synthesis and Translational Control
228
5-fluorouracil treatment of colorectal cancer cells alters ribosome biogenesis
and cytoplasmic ribosomal RNA composition
Gabriel Therizols1, Frederic Catez1, Coralie Carron2, Jérôme Guitton3, Zeina Bash Imam4,
Florian Laforêts4, Virginie Marcel4, Sabine Hacot4, Marie-Alexandra Albaret1, Hichem C
Mertani4, Jean-Christophe Saurin4, Jean-Jacques Diaz4
1 Cancer Research Center of Lyon, France
2 Virologie et pathologies humaines "Virpath », Faculté de médecine Laënnec, France
3 Laboratoire de toxicologie, Faculté de pharmacie de Lyon, France
4 Cancer Research Center of Lyon, Inserm U1052 CNRS 5286 UCBL, France
Presenter: Gabriel Therizols
It appears more and more clearly that the increase of ribosome production of cancer cells is
accompanied by alterations of their quality, leading to a modification of their function.
Ribosomes are ribonucleoprotein complexes responsible for protein synthesis. Structural
studies have revealed that ribosomal RNAs (rRNAs) play a major role in ribosome function
since they are the main support of the peptide bond formation and mRNA decoding. rRNAs
are chemically modified by 2'-O-methylations and pseudouridylations. These chemical
modifications participate to rRNA organization and to ribosome function. Alterations of rRNA
modifications affect translational fidelity and initiation modalities. 5-Fluorouracil (5-FU) is an
anti-metabolic drug used to treat many types of cancers. 5-FU was initially shown to affect
DNA metabolism through inhibition of thymidylate synthase. Several recent studies show that
the cytotoxic effect of 5-FU could indeed be due to its ability to be incorporated into RNA. This
incorporation affects the processing of a variety of RNA species including rRNA although the
functional consequences of 5-FU incorporation within rRNA remain to be fully elucidated. In
this work we have analysed the effect of 5-FU on ribosome biogenesis in the colorectal cancer
cell line HCT116. We show that 5-FU induces a modification of nucleolus ultrastructure with a
redistribution of several ribosome biogenesis factors. 5-FU treatment induces an accumulation
of pre-rRNA species and degradation of pre-28S resulting to a decrease of cytoplasmic
ribosome amount. However a small fraction of ribosomes are still exported into the cytoplasm
during the treatment. These ribosomes contain 5-FU within their rRNA and exhibit a modified
methylation patterns. From this observation, it can be anticipated that the function of these
ribosomes is altered. This work highlights a new aspect of cell-response to 5-FU treatment.
Work is ongoing to characterize the function of these modified ribosomes.
Page 264
Poster Abstracts
229
Selective mRNA translation in erythropoiesis
Klaske A.M.H. Thiadens1, Eleonora de Klerk2, Andrzej Nieradka3, Peter A.C. 't Hoen2,
Marieke von Lindern4
1 Sanquin Research Amsterdam, The Netherlands
2 Leiden University Medical Center, The Netherlands
3 ErasmusMC, Rotterdam, The Netherlands
4 Sanquin Research and Landsteiner Lab. AMC/UvA, The Netherlands
Presenter: Klaske A.M.H. Thiadens
Erythropoiesis requires a tightly controlled balance between expansion of the erythroblast
compartment and differentiation into mature erythrocytes, which is regulated by environmental
factors. Selective mRNA translational is an important mechanism in this control. The aim of our
research is to understand the molecular mechanisms involved in selective mRNA translation.
Expression profiling of polyribosomal and total mRNA identified multiple transcript whose
translation was hypersensitive to PI3K activation and eIF4E availability. One of the transcripts
subject to selective translation was Use1 (unusual SNARE in the ER) encoding an ER resident
SNARE protein involved in retrograde transport of cargo receptors and chaperones from the
Golgi back to the ER. In addition to structures that render translation hypersensitive to eIF4E,
the 5’UTR of Use1 contains several uORFs that regulate the translation initiation and the
synthesis of both a 31kDa protein, and a 21kDa isoform that lacks the N-terminus. This
regulation depended on the phosphorylation of eIF2. Not only the 5’UTR of Use1, also the
5´UTR of other transcripts that were identified as being hypersensitive to SCF-induced PI3K
activation and eIF4E availability contained uORFs. To detect which uORFs are translated and
may coordinate the expression of isoforms from Use1 and other transcripts, we use ribosome
profiling. In our experimental design we make use of the drugs cycloheximide and
harringtonine that arrest translation by either blocking elongation or blocking the ribosome at
the start site. We expect that ribosome profiling under different conditions, including
erythroblast proliferation and differentiation, and eIF2 phosphorylation, will give insight in the
regulation of Use1. It will also permit a genome wide analysis of protein isoforms expressed in
presence or absence of stress conditions that result in phosphorylation of eIF2.
Page 265
EMBO Conference Series: Protein Synthesis and Translational Control
230
Analyses of the translation termination factors in S. cerevisiae
Bettina Tieg, Simon Uhse, Alexandra Hackmann, Heike Krebber
Georg-August-University Göttingen, Germany
Presenter: Bettina Tieg, Simon Uhse
Upon synthesis and multiple assembly steps in the nucleus, the eukaryotic pre-ribosomal
particles are exported through the nuclear pore complex into the cytoplasm. Further
cytoplasmic maturation steps follow leading to ribosomal subunits competent for translation.
Upon translation initiation, elongation and subsequent arrival of the ribosomes at the stop
codon, translation termination is mediated by the eukaryotic release factors eRF1 and eRF3.
The termination codon in the ribosomal A-site is recognized by eRF1 (in S. cerevisiae encoded
by SUP45), which subsequently mediates the hydrolysis of the peptidyl-tRNA in the peptidyl
transferase center and the release of the polypeptide chain. The GTPase eRF3 (in S. cerevisiae
encoded by SUP35) stimulates the activity of eRF1 by GTP hydrolysis. In S. cerevisiae,
additional factors involved in translation termination were identified: The DEAD-box RNA
helicase Dbp5/Rat8 (Gross et al. 2007) and its stimulating factor Gle1 (Bolger et al. 2008),
which are well known for their function in the nuclear export of mRNAs. Furthermore, the
iron-sulfur containing ABC-family ATPase Rli1 was discovered (Khoshnevis et al. 2010).
Genetic and physical interactions with the canonical termination factors eRF1 and eRF3 were
identified and mutant strains reveal defects in the stop codons recognition. However, how
these factors function in translation termination and in which order they associate with the
termination complex is rather nebulous. One aim of our studies is to answer these questions
with in vivo and in vitro studies and to establish a new model for translation termination that
includes all factors.
Page 266
Poster Abstracts
231
Regulation of dendritogenesis by ZBP1 depends on its phosphorylation
at Ser181
Anna Urbanska, Jacek Jaworski
International Institute of Molecular and Cell Biology in Warsaw, Poland
Presenter: Anna Urbanska
Zipcode binding protein 1 (ZBP1) is one of several RNA binding proteins found in
ribonucleoparticles (RNPs) and dendritic processing bodies (P-bodies), structures involved in
mRNA silencing and transport - both required for local protein synthesis, a mechanism present
in various types of polarized cells. In neurons, local protein synthesis enables proper axonal
growth cone and spine formation, as well as appropriate dendritic arborization - features that
define electrical properties of a neuron. Recently we showed that dendritogenesis relies on
ZBP1-dependent dendritic transport of β-actin mRNA and its local translation. We also proved
that phosphorylation of ZBP1 by Src kinase is important for this process. Now we demonstrate
that ZBP1 is effectively phosphorylated in vitro by mTOR kinase. We took advantage of
recently published information regarding potential mTOR-dependent phosphorylation sites in
ZBP1 i.e. Ser181 (Dai et al., 2011), and examined role of this phosphorylation in ( i ) dendritic
arborization and ( ii ) cellular distribution of ZBP1. To address these questions we constructed
non-phosphorable (S181A) and phospho-mimicking (S181E) mutants of ZBP1 fused to GFP.
We observed that S181E, but not S181A reversed morphological deficits caused by ZBP1
knockdown. Another observation was that distribution along the dendrites of
non-phosphorable mutants was more even than the distribution of wild type ZBP1, which is
denser at the dendritic branching points. Thus we concluded that Ser181 phosphorylation is
involved in ZBP1 functions during dendritic growth. This research has been supported by
National Science Center (NCN) grant 2011/01/N/NZ3/05405
Page 267
EMBO Conference Series: Protein Synthesis and Translational Control
232
Translation elongation controls translation initiation on eukaryotic mRNAs
Tobias von der Haar
University of Kent, United Kingdom
Presenter: Tobias von der Haar
Synonymous codons encode the same amino acid, but differ in other biophysical properties.
These differences underpin the evolutionary selection of preferred codons. Recent studies
have shown that changing the proportion of preferred codons strongly affects protein
expression levels, and that natural codon usage controls biological processes from stress
resistance to circadian rhythms. However, no translational control mechanism has ever been
experimentally established that could link codon usage to protein expression levels. We now
demonstrate a novel translational control mechanism that responds to the speed of ribosome
movement around the start codon. High initiation rates are only possible if start codons are
liberated sufficiently fast, thus accounting for the observation that fast codons are
over-represented in highly expressed proteins. In contrast, slow codons lead to slow liberation
of the start codon by initiating ribosomes and interfere with efficient translation initiation.
Importantly, we show that physiological translation initiation and elongation rates are balanced
such that control can be transferred from initiation to elongation and vice versa via relatively
small changes in either process, and we demonstrate that translation elongation controls
protein expression levels from natural eukaryotic mRNAs. Our findings are not compatible with
recent interpretations of ribosome footprinting data that all codons are decoded with the same
speed in vivo. Instead, they implicate ribosomal speed control as a rich source of gene
expression regulation in eukaryotes.
Page 268
Poster Abstracts
233
Structural basis of signal sequence surveillance and selection by the SRP–FtsY
complex
Ottilie von Loeffelholz1, Kèvin Knoops1, Aileen Ariosa2, Xin Zhang2, Manikandan
Karuppasamy1, Karine Huard1, Guy Schoehn3, Imre Berger1, Shu-ou Shan2, Christiane
Schaffitzel1
1 EMBL Grenoble, France
2 California Institute of Technology, United States of America
3 Institute Biologie Structurale, France
Presenter: Ottilie von Loeffelholz
Signal-recognition particle (SRP)-dependent targeting of translating ribosomes to membranes
is a multistep quality-control process. Ribosomes that are translating weakly hydrophobic
signal sequences can be rejected from the targeting reaction even after they are bound to the
SRP. Here we show that the early complex, formed by Escherichia coli SRP and its receptor
FtsY with ribosomes translating the incorrect cargo EspP, is unstable and rearranges
inefficiently into subsequent conformational states, such that FtsY dissociation is favored over
successful targeting. The N-terminal extension of EspP is responsible for these defects in the
early targeting complex. The cryo-electron microscopy structure of this 'false' early complex
with EspP revealed an ordered M domain of SRP protein Ffh making two ribosomal contacts,
and the NG domains of Ffh and FtsY forming a distorted, flexible heterodimer. Our results
provide a structural basis for SRP-mediated signal-sequence selection during recruitment of
the SRP receptor.
Page 269
EMBO Conference Series: Protein Synthesis and Translational Control
234
Cap-independent translation of mRNAs encoded by yeast linear plasmids
Vaclav Vopalensky, Martin Pospisek
Charles University in Prague, Czech Republic
Presenter: Vaclav Vopalensky
Linear plasmids were found in a number of yeast species belonging to nine genera. The
genetic organization of yeast linear plasmids appears to be quite uniform with the most
thoroughly studied plasmids pGKL1 and pGKL2 from the yeast Kluyveromyces lactis. The
pGKL plasmids are peculiar in many respects since both plasmids are cytoplasmically
localized, linear and with distinct proteins covalently linked to their terminal inverted repeats.
Here we present molecular and functional analyses of UTRs from the plasmid-specific mRNAs.
We performed a molecular analysis of a putative capping enzyme encoded by K2ORF3 from
pGKL2. We produced K2Orf3p as a GST-tagged fusion protein in a E. coli expression system,
purified K2Orf3p by affinity chromatography and successfully tested for its guanylyltransferase
activity. Surprisingly, we were not able to detect any N7-methyltransferase activities of the
purified K2Orf3p protein. This result is further supported by our finding that pGKL mRNAs do
not bind to the cap-binding eukaryotic translation initiation factor 4E in vitro while cellular
mRNAs do and that a killer toxin, naturally encoded by the pGKL plasmids, is translated
independently of eIF4E in vivo, while control killer toxin gene artificially expressed under the
control of the strong Pol II driven promoter is translated by cap-dependent pathway. Last but
not least, we analyzed 3’ UTRs of the plasmid mRNAs and revealed that they are not
polyadenylated.
Page 270
Poster Abstracts
235
uORFdb – a comprehensive literature database on uORF biology
Klaus Wethmar1, Miguel A. Andrade-Navarro1, Adriano Barbosa-Silva2, Achim Leutz1
1 Max-Delbrueck-Center for Molecular Medicine, Germany
2 Luxembourg Centre for Systems Biomedicine, Luxembourg
Presenter: Klaus Wethmar
Approximately half of all human transcripts contain at least one upstream translational initiation
site that precedes the main coding sequence (CDS) and gives rise to an upstream open
reading frame (uORF). Upstream ORFs affect initiation rates at the CDS by interfering with
unrestrained progression of ribosomes across the 5´-transcript leader sequence. Although the
first uORF-related translational activity was noticed more than 30 years ago, only recently,
ribosome profiling and a growing list of physiological and medical implications attributed an
increased level of biological significance to uORF-mediated translational control. Here we
provide a comprehensive literature database on eukaryotic uORF biology (uORFdb). The
uORFdb categorizes individual uORF-related publications by a variety of denominators,
including species, gene, and the type of study. Most importantly, users can filter the database
for multiple structural and functional uORF-related properties that were manually identified in
the cited literature.
A web interface, publicly available at http://cbdm.mdc-berlin.de/tools/uorfdb/, allows
convenient and targeted access to information covering the complex field of uORF biology.
Page 271
EMBO Conference Series: Protein Synthesis and Translational Control
236
Identification of conserved intramolecular communication pathways
within EF-Tu.
Hans-Joachim Wieden, Evan Mercier, Fan Mo, Dylan Girodat
University of Lethbridge, Canada
Presenter: Hans-Joachim Wieden
During the elongation cycle of bacterial protein synthesis elongation factor (EF) Tu delivers
aminoacyl(aa)- tRNAs to the ribosome in a GTP-dependent manner. As a checkpoint for
correct codon-anticodon interaction EF-Tu has to integrate a diverse set of input signals. In
order to study how this signal integration is achieved communication processes within the
protein have to be analyzed. Here we report the construction of an intramolecular
communication network for EF-Tu based on a series of molecular dynamics (MD) simulations,
representing the dynamic properties of EF-Tu as a complex set of long-range signal
transmission pathways. We identify conserved communication pathways important for GTPase
activation on the ribosome as well as EF-Ts-catalyzed nucleotide exchange. Single amino acid
substitutions in EF-Tu can change the network organization dramatically and result in
significantly reduced communication between domains across a set of universally conserved
interdomain bridges. To validate our findings in vitro we have performed Michaelis-Menten
analyses to study the ribosome stimulated GTP hydrolysis activity of EF-Tu. In order to isolate
this signal pathway from any aa-tRNA contributions we performed these experiments in the
absence of aa-tRNA. Consistent with our network analysis, we find that single amino acid
substitutions in domain II reduce the stimulatory effect of the 70S ribosome by five-fold, while
not affecting 50S-dependent stimulation. Furthermore, variants of EF-Tu found to disrupt the
conserved interdomain bridges reduce the stimulatory effect of the 70S ribosome to a similar
extent. Analysis of an intramolecular communication network constructed for the EF-Tu•EF-Ts
binary complex revealed an independent set of communication pathways promoting nucleotide
exchange in EF-Tu. The role of key amino acids in this network was validated using
rapid-kinetics techniques to determine nucleotide-binding properties.
Page 272
Poster Abstracts
237
Dimerisation of the eIF2B complex, a potential new mechanism for translational
control?
Noel Wortham1, Magdalena Martinez1, Yuliya Godiyenko2, Carol Robinson2, Christopher
Proud1
1 University of Southampton, United Kingdom
2 University of Oxford, United Kingdom
Presenter: Noel Wortham
The translation initiation factor eIF2B is a key regulator of translation initiation through its action
as the guanine nucleotide exchange factor (GEF) for eIF2. Unusually for a GEF, eIF2B is a
multimeric factor comprising 5 subunits, α - ε. eIF2B has traditionally been regarded as a
pentamer. However, the recent crystal structure of eIF2Bα and the structures of homologous
proteins suggest that this subunit may dimerise. We have shown this is indeed the case and,
furthermore, have demonstrated that the complete mammalian eIF2B complex exists as a
decamer (a dimer of pentamers, ( a b g d e ) 2 ). eIF2B complexes lacking eIF2Bα are unable
to form this structure and instead form tetrameric b g d e complexes. It has previously been
shown that eIF2Bα is required for inhibition of eIF2B by phosphorylated eIF2, which is
increased in response to many cellular stresses. Furthermore, we have shown that eIF2B
complexes lacking eIF2Bα are only half as active as full complexes, suggesting that
dimerisation may be required for full activity of the complex. Purification of native eIF2B from
cytoplasmic extracts suggests that both full eIF2B complexes and complexes lacking eIF2Bα
are normally present in the cell. These data have implications for the regulation of both eIF2B
activity and cellular responses to stress. Complexes containing eIF2Bα are more active, with
the caveat of increased susceptibility to inhibition by eIF2 phosphorylation, whereas complexes
lacking eIF2Bα are less active, but are insensitive to inhibition by phosphorylated eIF2. Thus,
the cellular level of eIF2Bα appears likely to determine the sensitivity of a cell’s response to
eIF2 phosphorylation, modulating its response to stress. This change in cell-specific response
to eIF2 phosphorylation resulting from differing expression levels of eIF2Bα may be important in
the pathogenesis of Vanishing White Matter (VWM), which is caused by mutations in the genes
encoding the eIF2B subunits.
Page 273
EMBO Conference Series: Protein Synthesis and Translational Control
238
Translational control of IFN-beta mRNA via RNA-binding proteins
Akiko Yanagiya, Tommy Alain, Nahum Sonenberg
McGill University, Canada
Presenter: Akiko Yanagiya
Though innate immune response is essential to defend cells, it is a double-edged sword since
excessive inflammatory response damages cells. Post-transcriptional mechanism has emerged
as a key player in control of innate immune response because of its rapid and flexible
regulation. Though post-transcriptional mechanisms dampen the expression of inflammatory
mediators by mRNA decay, little is known about translational control of their mRNAs. Many
transcripts encoding immune regulatory proteins possess a variety of cis elements in 3’UTR,
and their mRNA decay is mediated by coordinated association between cis elements and
RNA-binding proteins. The transient production of IFN-β is triggered in response to viral
infection or poly (I:C). Post-transcriptional control plays a key role in IFN-β production since
translation of IFN-β mRNA with a long poly(A) tail is inhibited, whereas that with a short poly(A)
tail is translated efficiently. IFN-β mRNA contains AREs in 3’UTR, which affect translational
efficiency and mRNA stability. Though AREs regulate translation of IFN-β mRNA by interacting
with poly(A) tail, little is known about translational regulation of IFN-β mRNA via ARE- and
poly(A) tail-dependent mechanisms. P oly(A)- b inding p rotein (PABP) plays a pivotal role in
mRNA stabilization and translation, and PAIP2A suppresses its activity by releasing PABP from
poly(A) tail. Intriguingly, IFN-β production induced by poly(I:C) treatment was dramatically
inhibited in PAIP2A-KO MEFs, suggesting a key role of PAIP2A in translational regulation of
IFN-β mRNA. Proteomic analysis and binding assay using recombinant PAIP2A reveals that
PAIP2A directly binds to RNA-binding proteins such as hnRNPs and ARE-binding protein,
HuR. We address translational control of IFN-β mRNA via cis elements such as ARE and
poly(A) tail, and RNA-binding proteins which associate with PAIP2A such as PABP, HuR and
hnRNPs.
Page 274
Poster Abstracts
239
Regulation and function of Rps6 Phosphorylation in budding yeast
Seda Yerlikaya, Madeleine Meusburger, Robbie Loewith
University of Geneva, Switzerland
Presenter: Seda Yerlikaya
For the past 30 years, the function of the nutrient-regulated phosphorylation of ribosomal
protein S6 has remained mysterious. Probing this problem in the model eukaryote
Saccharomyces cerevisiae we found that phosphorylation of the two phosphosites in yeast
Rps6 are differentially regulated downstream of Target Of Rapamycin Complexes 1 and 2.
TORC1 regulates phosphorylation of both sites directly via the poorly characterized AGC-family
kinase Ypk3, and indirectly via Sch9 and its control on mRNA translation. TORC2 regulates
phosphorylation of only the N-terminal phosphosite via Ypk1 and Ypk2. Glc7, the yeast PP1
phosphatase mediates Rps6 dephosphorylation. Strains harbouring phosphomimetic and
non-phosphorylatable variants of Rps6 have scorable phenotypes but do not phenocopy
Rps6-kinase mutants suggesting that these are poor models to define functional roles for Rps6
phosphorylation.
Page 275
EMBO Conference Series: Protein Synthesis and Translational Control
240
DAP5 mediated translation control of human embryonic stem cell
differentiation
Yael Yoffe, Rinat Kalaora, Adi Kimchi
Weizmann Institute of Science, Israel
Presenter: Yael Yoffe
Little is known about the translational mechanisms that regulate embryonic stem cells (ESCs)
pluripotency and differentiation. DAP5 is an eIF4G homologue that mediates cap-independent
translation, by serving as a scaffold for pre initiation complex (PIC) assembly at Internal
Ribosome Entry Sites (IRES). Until now, this alternate form of translation initiation has been
investigated mainly under cell stress and apoptotic conditions when cap dependent translation
is compromised. Notably, it has been reported that DAP5 knock-out mice die at an early stage
of gastrulation, and that the differentiation of DAP5 -/- mouse ESCs is impaired. This most
likely stems from a critical DAP5 function in mediating IRES-dependent translation of specific
target mRNAs that are necessary for differentiation of ESCs. To test this hypothesis, we have
generated stable human ESC expressing control or DAP5-targeting shRNA to successfully
knock-down (KD) DAP5 expression. DAP5 KD cells showed normal hESC morphology.
However, DAP5 had a critical role in differentiation of the hESCs under retinoic acid (RA)
treatment and spontaneous differentiation as embryoid bodies (EB). DAP5 depletion prevented
very significantly the strong reduction in the expression of the pluripotent markers Nanog and
Oct4 in 3-20 days EBs as compared to control cells. At the phenotypic level, DAP5 KD EBs
failed to undergo cavitation and differentiation. Also, DAP5 KD EBs showed enhanced
non-localized apoptosis throughout the EBs. These data suggest that DAP5 is critical for the
differentiation of hESCs. The global translation profile of pluripotent DAP5 KD cells has no
major difference from control cells. However, using deep sequencing of RNA from heavy
polysomal fractions we identified potential mRNA targets of DAP5 translation which are
currently being analyzed at the functional level.
Page 276
Poster Abstracts
241
eIF1 phosphorylation mediates leaky scanning translation initiation
Lolita Zach
Technion- institute of technology, Israel
Presenter: Lolita Zach
Accurate protein synthesis is required in order to establish acorrect proteome. Initial steps in
protein synthesis are highly regulated processes as they define the reading frame of translation
and commit the translation machinery to begin the elongation phase. Eukaryotic translation
initiation is a process facilitated by numerous factors (eIFs), aimed to form a “scanning”
mechanism to the initiation codon. Translation initiation has been reported to be regulated by
re-initiation and leaky scanning. Re-initiation regulation is governed by the phosphorylation
status of eIF2alpha and controlled by cellular stresses via the Integrated Stress Response (ISR)
pathway. However, regulation of the leaky scanning translation initiation is not known to be
regulated or connected to cellular conditions. We have identified AIRAP to be translationally
induced in a leaky scanning regulatory manner. AIRAP transcript contains a single upstream
Open Reading Frame (uORF) in a poor-kozak context. Translation induction during stress
conditions is governed by means of leaky scanning and not re-initiation. This induction of
AIRAP is solely dependent on eIF1 and the uORF kozak context. We show that eIF1 is
phosphorylated under specific conditions that induce protein misfolding and have
biochemically characterized this site of phosphorylation. Our data indicate that leaky scanning
like re-initiation is responsive to stress conditions and that leaky scanning can govern ORF
translation induction by bypassing poor kozak context of a single uORF transcript.
Page 277
EMBO Conference Series: Protein Synthesis and Translational Control
242
The role of Pet309 in mitochondrial translation initiation of the COX1 mRNA
Angelica Raquel Zamudio Ochoa, Yolanda Camacho Villasana, Aldo García Guerrero,
Xochitl Pérez Martínez
Instituto de Fisiología Celular, UNAM, Mexico
Presenter: Angelica Raquel Zamudio Ochoa
Mitochondrial translation initiation is not completely understood. Unlike bacteria, the
mitochondrial mRNAs do not contain a Shine-Dalgarno sequence, and it has been proven that
the scanning mechanism is not used in this organelle. In yeast, eight proteins are encoded in
the mitochondrial genome, and Cox1 is one of them. Cox1 is the largest subunit of Complex IV
of the respiratory chain. Its synthesis inside mitochondria is tightly regulated. Pet309 is a
nuclear encoded factor, which initiates translation of the COX1 mRNA acting on its 5’ UTR.
The exact mechanism of action of Pet309 is not known but its sequence predicts 23 PPR
motifs that could mediate interaction with the COX1 mRNA. Our hypothesis was that Pet309
interacts with the mRNA of COX1 and with the mitochondrial ribosome to locate it on the start
codon of the mRNA. To prove that Pet309 interacts with the mRNA of COX1, we
immunoprecipitated the activator with specific antibodies and purified the RNA of the
precipitate. By RT-PCR assays we identified the COX1 mRNA in this precipitate. To test if
there was an interaction of Pet309 with the mitoribosome we ran mitochondrial extracts on a
sucrose gradient. We observed a co-migration of Pet309 with ribosomal proteins. In a strain
that does not assembly the mitoribosome, Pet309 was detected on the top of the gradient.
Interestingly, the presence of the COX1 mRNA was not necessary for the interaction of Pet309
with the ribosome. Elimination of the first 6 PPR domains of Pet309 abolished the
co-migration. We conclude that Pet309 interacts with the COX1 mRNA. It also interacts with
the mitoribosome through its amino terminal region. The interaction with the ribosome seems
to be a direct rather than mediated by the COX1 mRNA. These data supports our hypothesis
in which Pet309 locates the ribosome on the start codon of the COX1 mRNA.
Page 278
Poster Abstracts
243
eIF5A has a function in the cotranslational translocation of proteins into the ER
Cleslei Zanelli, Danuza Rossi, Fabio Carrilho Galvão, Hermano Martins Bellato, Paulo
Boldrin, Brenda J Andrews, Sandro Roberto Valentini
Univ Estadual Paulista – UNESP, Brazil
Presenter: Cleslei Zanelli
The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved and
essential protein present in all organisms excepted in bacteria. eIF5A requires the modification
of a specific residue of lysine to hypusine for its activation. The hypusine modification occurs
posttranslationally in two steps and the polyamine spermidine is necessary for this
modification. Despite having an essential function in translation elongation, the critical role
played by eIF5A remains unclear. Besides demonstrating genetic interactions with translation
factors, eIF5A mutants genetically interact with mutations in YPT1, which encodes an essential
protein involved in RE-to-Golgi vesicle transport. Herein, we investigated the correlation
between eIF5A function in translation and secretion in yeast. Results of in vivo translocation
assays and genetic interactions analysis suggest a specific role for eIF5A in the cotranslational
translocation of proteins to he, but not in the posttranslational pathway. Additionally, we
observed that a block in eIF5A activation up-regulates stress-induced chaperones, which also
occurs when SRP function is lost. Finally, eIF5A was important for binding of the
ribosome-nascent chain complex to SRP. These results link eIF5A function in translation with
the role of SRP in the cell and may help explain the dual effects of eIF5A in differential and
general translation.
Page 279
EMBO Conference Series: Protein Synthesis and Translational Control
244
System-wide posttranscriptional responses of NIH-3T3 cells to genotoxic
stress
Elisabeth M Zielonka1, Anne-Marie Alleaume1, Sophia Foehr1, Bernd Fischer1, Alfredo
Castello1, Jeroen Krijgsveld1, Matthias Hentze1
1 EMBL Heidelberg, Germany
Presenter: Elisabeth M Zielonka
RNA binding proteins (RBP) play critical roles in stress responses to DNA damage through
interactions with mRNAs that encode functionally relevant proteins. One of the most cytotoxic
forms of DNA damage are DNA double strand breaks (DSB), which can cause mutagenic
events or cell death. This project focuses on the plasticity of the global RBP network (mRNA
interactome) and its target RNAs (RNA regulons) of mouse fibroblasts (NIH 3T3 cells) in
response to either sublethal or lethal doses of gamma irradiation. Using interactome capture
[1; 2] and high resolution proteomics, we defined the mRNA interactome of NIH 3T3 cells
under non stressed conditions. From over 490 identified RBP´s, which are significantly
enriched in the cross linked over the non cross linked samples, 169 had not previously been
implicated in aspects of RNA biology. Ongoing experiments will define the responses of
different RBPs of the interactome to genotoxic stress using quantitative mass spectrometry.
This will be followed up by functional assays to determine their influence on cell survival. Taken
together, this project aims to address key questions of the DNA damage response and the
implication of RNA regulons.
References:
[1] Castello A. et al., Cell 2012, 149 (6); [2] Castello A. et al., Nat Protoc. 2013, 8 (3)
Page 280
List of Participants
A
Florian Aeschimann
Friedrich Miescher Institute for Biomedical
Research
Switzerland
[email protected]
Tommy Alain
Mcgill University
Canada
[email protected]
Dmitry Andreev
Moscow State University
Russian Federation
[email protected]
Cammas Anne
INSERM
France
[email protected]
Sanja Antic
Max F. Perutz Laboratories & University of
Vienna
Austria
[email protected]
Stefan Arenz
Gene Center Munich, LMU Munich
Germany
[email protected]
John Atkins
University of Utah/University College Cork
United States of America
[email protected]
B
Eric Baggs
Department of Microbiology and Molecular
Genetics, University of California Irvine
United States of America
[email protected]
Nenad Ban
ETH Zurich
Switzerland
[email protected]
Pavel Baranov
University College Cork
Ireland
[email protected]
Jiri Bartek
Danish Cancer Society
Denmark
[email protected]
Amandine Bastide
MRC, Toxicology Unit
United Kingdom
[email protected]
Ira Bavli-Kertselli
Technion
Israel
[email protected]
Thomas Becker
Beckmann Group
Germany
[email protected]
Benedikt Beckmann
EMBL Heidelberg
Germany
[email protected]
Roland Beckmann
Gene Center Munich
Germany
[email protected]
Petra Beznoskova
Academy of Sciences of the Czech Republic
Czech Republic
[email protected]
Mamatha Bhat
McGill
Canada
[email protected]
Stefano Biffo
Fondazione Centro San Raffaele del Monte
Tabor & Università del Piemonte Orientale
Italy
[email protected]
Page 281
EMBO Conference Series: Protein Synthesis and Translational Control
Mark Biggin
Lawrence Berkeley National Laboratory
United States of America
[email protected]
Scott Blanchard
Cornell Med
United States of America
[email protected]
Sandra Blanchet
Institut de Genetique et Microbiologie
France
[email protected]
Christian Blau
MPI for Biophysical Chemistry
Germany
[email protected]
Elzbieta Bojarska
University of Warsaw
Poland
[email protected]
Harland Brandon
University of Lethbridge
Canada
[email protected]
Daniela Brina
San Raffaele Scientific institute
Italy
[email protected]
Matthew Brook
University of Edinburgh
United Kingdom
[email protected]
Mario Brosch
University Hospital Kiel
Germany
[email protected]
Dmitry Burakovskiy
MPI for Biophysical Chemistry
Germany
[email protected]
Martin Bushell
MRC Toxicology Unit
United Kingdom
[email protected]
C
Neva Caliskan
MPI for Biophysical Chemistry
Germany
[email protected]
Cornelis Calkhoven
Leibniz Institute for Age Research - Fritz
Lipmann Institute
Germany
[email protected]
Tavane Cambiaghi
Unifesp
Brazil
[email protected]
Mar Castellano
INIA
Spain
[email protected]
Lydia Castelli
University of Manchester
United Kingdom
[email protected]
Alfredo Castello
EMBL Heidelberg
Germany
[email protected]
Beatriz Castilho
UNIFESP
Brazil
[email protected]
Ana Beatriz Castro
INIA
Spain
[email protected]
Frederic Catez
Cancer Research Center of Lyon
France
[email protected]
Page 282
List of Participants
Regina Cencic
McGill University
Canada
[email protected]
Joanne Cowan
University of Southampton
United Kingdom
[email protected]
Tirtha Chakraborty
Moderna
United States of America
[email protected]
Joseph Curran
University of Geneva Medical School
Switzerland
[email protected]
Li-Kwan Chang
National Taiwan university
Taiwan
[email protected]
Guo-Liang Chew
Harvard University
United States of America
[email protected]
Nnaji Christiancia Ifeyinwa
National Biotechnology Development Agency
Nigeria
[email protected]
Matthew Cockman
University of Oxford
United Kingdom
[email protected]
Mark Coldwell
University of Southampton
United Kingdom
[email protected]
Mathew Coleman
University of Oxford
United Kingdom
[email protected]
Simon Cook
The Babraham Institute
United Kingdom
[email protected]
Amy Cooke
EMBL Heidelberg
Germany
[email protected]
D
Kim De Keersmaecker
K.U.Leuven - VIB
Belgium
[email protected]
Quentin Defenouillère
Institut Pasteur
France
[email protected]
Natalia Demeshkina
IGBMC
France
[email protected]
Guillaume Desnoyers
Atlantic Cancer Research Institute
Canada
[email protected]
Thomas Dever
NIH
United States of America
[email protected]
Rivka Dikstein
Weizmann Institute of Science
Israel
[email protected]
Sergey Dmitriev
Moscow State University
Russian Federation
[email protected]
Silke Dorner
Max F. Perutz Laboratories & University of
Vienna
Austria
[email protected]
Page 283
EMBO Conference Series: Protein Synthesis and Translational Control
Albena Draycheva
MPI for Biophysical Chemistry
Germany
[email protected]
Nuria Fernandez Bautista-Abad
UPM
Spain
[email protected]
Joshua Dunn
University of California San Francisco
United States of America
[email protected]
Sonia Fieulaine
Centre National de la Recherche Scientifique
Mélodie Duval
IBMC-CNRS
France
[email protected]
Susan Dwane
University of Limerick
Ireland
[email protected]
France
[email protected]
Andrew Firth
University of Cambridge
United Kingdom
[email protected]
Niels Fischer
MPI for Biophysical Chemistry
Germany
[email protected]
E
Andrew Easton
University of Warwick
United Kingdom
[email protected]
Orna Elroy-Stein
Tel Aviv University
Israel
[email protected]
Irina Epstein
Max Planck Institute for Brain Research
Germany
[email protected]
Katri Eskelin
University of Helsinki
Finland
[email protected]
F
Tianshu Feng
Oxford University
United Kingdom
[email protected]
Dominique Fourmy
CNRS
France
[email protected]
Paul Fox
Cleveland Clinic
United States of America
[email protected]
Andrew Friday
Brody School of Medicine
United States of America
[email protected]
Claudia Fritsch
University Hospital Kiel
Germany
[email protected]
Ludmila Frolova
Institute of Molecular Biology RAN
Russian Federation
[email protected]
Sigal Frost
Ben-Gurion University of the Negev
Israel
[email protected]
G
Page 284
List of Participants
Gabriela Galicia Vazquez
McGill University
Canada
[email protected]
Sebastian Glatt
EMBL Heidelberg
Germany
[email protected]
Magdalena Gamm
Universiteit Utrecht
The Netherlands
[email protected]
Agnieszka Gorska
Institute of Bioorganic Chemistry Polish
Academy of Sciences
Poland
[email protected]
Marina García-Beyaert
Centre for genomic regulation (CRG)
Spain
[email protected]
Nicolas Garreau de Loubresse
IGBMC
France
[email protected]
Daria Gawron
VIB
Belgium
[email protected]
Fátima Gebauer
Centre for Genomic Regulation (CRG)
Spain
[email protected]
Songsong Geng
McGill University
Canada
[email protected]
Robert Gilbert
University of Oxford
United Kingdom
[email protected]
Antonio Giraldez
Yale University
United States of America
[email protected]
Tiziana Girardi
KU Leuven
Belgium
[email protected]
Phillip Gould
University of Warwick
United Kingdom
[email protected]
Nicola Gray
University of Edinburgh
United Kingdom
[email protected]
Stefano Grosso
MRC, Toxicology Unit
United Kingdom
[email protected]
Renata Grzela
Centre National de la Recherche Scientifique
France
[email protected]
H
Katharina Haneke
DKFZ-ZMBH Alliance
Germany
[email protected]
Johannes Hanson
Utrecht University
The Netherlands
[email protected]
Christopher Hellen
State University of New York Downstate
Medical Center
United States of America
[email protected]
Page 285
EMBO Conference Series: Protein Synthesis and Translational Control
Matthias Hentze
EMBL Heidelberg
Germany
[email protected]
Ivaylo Ivanov
University College Cork
Ireland
[email protected]
Anna Herrmannová
Academy of Sciences of the Czech Republic
Czech Republic
[email protected]
Pavel Ivanov
Brigham nad Women's Hospital/Harvard
Medical School
United States of America
[email protected]
Hans Heus
Radboud University Nijmegen
The Netherlands
[email protected]
Alan Hinnebusch
National Institutes of Health
United States of America
[email protected]
Isabel Hofman
KU Leuven
Belgium
[email protected]
Stephen Hoge
Moderna
United States of America
[email protected]
Wolf Hagen Holtkamp
MPI for biophysical Chemistry
Germany
[email protected]
Rastislav Horos
EMBL Heidelberg
Germany
[email protected]
I
Valentina Iadevaia
University of Southampton
United Kingdom
[email protected]
Elena Ivanova
University of Geneva
Switzerland
[email protected]
Elisa Izaurralde
MPI for Developmental Biology
Germany
[email protected]
J
Richard Jackson
University of Cambridge
United Kingdom
[email protected]
Seyed Mehdi Jafarnejad
McGill University
Canada
[email protected]
Sung Key Jang
POSTECH
Korea, Republic of
[email protected]
Peggy Janich
University of Lausanne
Switzerland
[email protected]
Martin Jennings
University of Manchester
United Kingdom
[email protected]
Nicholas Ingolia
Carnegie Institution for Science
United States of America
[email protected]
K
Page 286
List of Participants
Helena Kaija
University of Oulu
Finland
[email protected]
Susanne Kramer
Würzburg University
Germany
[email protected]
Anastasiia Kamenska
University of Cambridge
United Kingdom
[email protected]
Dorota Kubacka
University of Warsaw
Poland
[email protected]
Brett Keiper
Brody School of Medicine at East Carolina
University
United States of America
[email protected]
Stefan Kubick
Fraunhofer Institute for Biomedical Engineering
Chris Kershaw
University of Manchester
United Kingdom
[email protected]
Pavel Kudrin
Tartu University
Estonia
[email protected]
Debjit Khan
Indian Institute of Science
India
[email protected]
Scott Kuersten
Epicentre (An Illumina Company)
United States of America
[email protected]
Konstantin Khetchoumian
IRCM
Canada
[email protected]
Bernhard Kuhle
Georg-August-University Göttingen
Germany
[email protected]
Michael Kiebler
LMU Munich
Germany
[email protected]
Yuichi Kimura
University of Tsukuba
Japan
[email protected]
John Knight
MRC Toxicology Unit
United Kingdom
[email protected]
Suresh Kotini
MPI for Biophysical Chemistry
Germany
[email protected]
Germany
[email protected]
L
Rafaela Lacerda
Instituto Nacional de Saúde Dr.Ricardo Jorge
Portugal
[email protected]
Charline Lasfargues
INSERM CRCT
Germany
[email protected]
Jeroen Lastdrager
Utrecht University
The Netherlands
[email protected]
Page 287
EMBO Conference Series: Protein Synthesis and Translational Control
Sejeong Lee
MPI for Biophysical Chemistry
Germany
[email protected]
Seung Hwan Lee
POSTECH
Korea, Republic of
[email protected]
Julia Legen
Humboldt University Berlin
Germany
[email protected]
Anja Lehweß-Litzmann
MPI for Biophysical Chemistry
Germany
[email protected]
Sebastian Leidel
MPI for Molecular Biomedicine
Germany
[email protected]
Anke Liepelt
University Hospital, RWTH Aachen
Germany
[email protected]
Lisa Lindqvist
Walter & Eliza Hall Institute of Medical
Research
Australia
[email protected]
Yi Liu
Göttingen University
Germany
[email protected]
Ying Liu
University of Helsinki
Finland
[email protected]
Ivan Lomakin
Yale University
United States of America
[email protected]
Marcelo Lopez-Lastra
Escuela de Medicina, Pontificia Universidad
Católica de Chile
Chile
[email protected]
Fabrizio Loreni
University of Rome Tor Vergata
Italy
[email protected]
Tetyana Lukash
Institute of molecular biology and genetics
Ukraine
[email protected]
Maciej Lukaszewicz
University of Warsaw
Poland
[email protected]
M
Paul Macdonald
University of Texas at Austin
United States of America
[email protected]
Justine Mailliot
IGBMC
France
[email protected]
Giovanna Mallucci
MRC Toxicology Unit
United Kingdom
[email protected]
Naglis Malys
University of Warwick
United Kingdom
[email protected]
Eder Mancera-Martinez
Institut de Biologie Moléculaire de Plantes
France
[email protected]
Virginie Marcel
Cancer Research Center of Lyon
France
[email protected]
Page 288
List of Participants
Ana Marques-Ramos
Instituto Nacional de Saúde Dr. Ricardo Jorge
Portugal
[email protected]
Franck Martin
Institut de Biologie Moléculaire et Cellulaire
France
[email protected]
Yvan Martineau
Cancer Research Center of Toulouse
France
[email protected]
Audrey Michel
University College Cork
Ireland
[email protected]
Marija Mihailovic
European Institute of Oncology
Italy
[email protected]
Tatiana Mikhaylova
Engelhardt Institute of Molecular Biology RAS
Russian Federation
[email protected]
Encarna Martinez-Salas
Centro de Biologia Molecular
Spain
[email protected]
Stefania Millevoi
INSERM
France
[email protected]
Juan Mata
University of Cambridge
United Kingdom
[email protected]
Eric Mills
Johns Hopkins University School of Medicine
Anna McGeachy
Johns Hopkins
United States of America
[email protected]
Yves Mechulam
Ecole Polytechnique-CNRS
France
[email protected]
Xiang Meng
University of Warwick
United Kingdom
[email protected]
William Merrick
Case Western Reserve University
United States of America
[email protected]
Oded Meyuhas
The Hebrew University-Hadassah Medical
School
Israel
[email protected]
United States of America
[email protected]
Angelika Modelska
University of Trento
Italy
[email protected]
Abdelrhman Mohamed
Dalgroup
Sudan
[email protected]
Sarah Mohammad-Qureshi
University of Manchester
United Kingdom
[email protected]
Andrew Moore
Wiley-VCH Verlag GmbH & Co.KGaA
Germany
[email protected]
Kat Moore
Sanquin Research
The Netherlands
[email protected]
Page 289
EMBO Conference Series: Protein Synthesis and Translational Control
J. Kaitlin Morrison
Brody School of Medicine at East Carolina
University
United States of America
[email protected]
Pamela Nicholson
University of Bern
Switzerland
[email protected]
Oliver Mühlemann
University of Bern
Switzerland
[email protected]
Michael Niepmann
Justus-Liebig-University
Germany
[email protected]
n.de
David Müller
INSERM
France
[email protected]
Benedikt Nilges
MPI for Molecular Biomedicine
Germany
[email protected]
Antonio Munoz
Johns Hopkins University School of Medicine
United States of America
[email protected]
Paola Nocua
Pontificia Universidad Javeriana Bogotá Colombia
Colombia
[email protected]
Sergey Mureev
The University of Queensland
Australia
[email protected]
Jonas Noeske
University of California at Berkeley
United States of America
[email protected]
N
Sabarish Nagarajan
University of Calgary
Canada
[email protected]
Olivier Namy
CNRS
France
[email protected]
Marco Nousch
MPI-CBG
Germany
[email protected]
Eva Maria Novoa
Institute for Research in Biomedicine
Spain
[email protected]
O
Kripa Nand
Talwar Research Foundation
India
[email protected]
Edward O'Brien
University of Cambridge
United Kingdom
[email protected]
Danny Nedialkova
MPI for Molecular Biomedicine
Germany
[email protected]
Theo Ohlmann
INSERM-ENS de LYON
France
[email protected]
Page 290
List of Participants
René Olsthoorn
Leiden University
The Netherlands
[email protected]
Lisa Perry
University of Southampton
United Kingdom
[email protected]
Dirk Ostareck
University Hospital RTWH Aachen
Germany
[email protected]
Tatyana Pestova
SUNY Downstate Medical Center
United States of America
[email protected]
Antje Ostareck-Lederer
University Hospital RWTH Aachen
Germany
[email protected]
Xavier Pichon
MRC Toxicology unit
United Kingdom
[email protected]
Ilya Osterman
Lomonosov Moscow State University
Russian Federation
[email protected]
Ramesh Pillai
EMBL Grenoble
France
[email protected]
P
Poonam Pandey
NCCS
India
[email protected]
Baptiste Panthu
CIRI
France
[email protected]
Luigi Pasini
Centre for Integrative Biology (CIBIO)
Italy
[email protected]
Graham Pavitt
University of Manchester
United Kingdom
[email protected]
Simone Pellegrino
IGBMC
France
[email protected]
Sandro Pereira
Columbia University
United States of America
[email protected]
David Piñeiro
MRC, Toxicology Unit
United Kingdom
[email protected]
Yves Poirier
University of Lausanne
Switzerland
[email protected]
Vitaly Polunovsky
University of Minnesota
United States of America
[email protected]
Katja Porvari
University of Oulu
Finland
[email protected]
Martin Pospisek
Charles University
Czech Republic
[email protected]
Anne Preis
LMU Munich Genecenter
Germany
[email protected]
Page 291
EMBO Conference Series: Protein Synthesis and Translational Control
Thomas Preiss
The Australian National University
Australia
[email protected]
Laura Ranum
University of Florida
United States of America
[email protected]
Irina Prokhorova
IGBMC
France
[email protected]
Partho Sarothi Ray
Indian Institute of Science Education and
Research
India
[email protected]
Christopher Proud
University of Southampton
United Kingdom
[email protected]
Alessandro Provenzani
CIBIO- University of Trento
Italy
[email protected]
Stephane Pyronnet
INSERM U858
France
[email protected]
Q
Alessandro Quattrone
University of Trento
Italy
[email protected]
R
Aditya Radhakrishnan
Johns Hopkins Medical Institute
United States of America
[email protected]
Marek Rajman
University of Marburg
Germany
[email protected]
Santiago Ramon y Cajal
Hospital Universitario Vall d'Hebron
Spain
[email protected]
Sariri Reyhaneh
University of Guilan
Iran
[email protected]
Marina Rodnina
MPI for Biophysical Chemistry
Germany
[email protected]
Luísa Romão
Inst. Nacional Saúde Dr. Ricardo Jorge
Portugal
[email protected]
Joanna Rorbach
MRC MBU
United Kingdom
[email protected]
Danuza Rossi
UNESP
Brazil
[email protected]
Alexey Rozov
IGBMC
France
[email protected]
Benedetta Ruzzenente
Max-Planck Institute for Biology of Ageing
Germany
[email protected]
S
Matthieu Saguy
Université Paris Sud
France
[email protected]
Page 292
List of Participants
Karissa Sanbonmatsu
Los Alamos National Laboratory and New
Mexico Consortium
United States of America
[email protected]
Karine Santos
Free University Berlin
Germany
[email protected]
Evelyn Sattlegger
Massey University
New Zealand
[email protected]
Anca Savulescu
CSIR
South Africa
[email protected]
Manisha Saxena
University of Bern
Switzerland
[email protected]
Christiane Schaffitzel
EMBL Grenoble
France
[email protected]
Claudia Scheckel
Rockefeller University
United States of America
[email protected]
Johanna Schott
German Cancer Research Center
Germany
[email protected]
Julia Schulz
Max Delbrück Center for Molecular Medicine
Germany
[email protected]
Birgit Schuster
EMBL Heidelberg
Germany
[email protected]
Juliane Schwarz
MPI for Molecular Biomedicine
Germany
[email protected]
Rok Sekirnik
University of Oxford
United Kingdom
[email protected]
Michal Shapira
Ben Gurion University of the Negev
Israel
[email protected]
Mikhail Shchepetilnikov
Université de Strasbourg
France
[email protected]
Daniela Schibich
DKFZ-ZMBH Alliance
Germany
[email protected]
Viktoryia Sidarovich
CIBIO- University of Trento
Italy
[email protected]
Tobias Schmid
Goethe-University Frankfurt
Germany
[email protected]
Angelita Simonetti
IGBMC
France
[email protected]
James Schofield
University of Southampton
United Kingdom
[email protected]
Clare Simpson
The University of Cambridge
United Kingdom
[email protected]
Page 293
EMBO Conference Series: Protein Synthesis and Translational Control
Hadar Sinvani
weizmann institute
Israel
[email protected]
Ariel Stanhill
Technion
Israel
[email protected]
Victoria Smirnova
Lomonosov Moscow State University
Russian Federation
[email protected]
Agata Starosta
Gene Center Munich, LMU Munich
Germany
[email protected]
Ewan Smith
MRC Toxicology Unit
United Kingdom
[email protected]
Joanna Stewart
University of Southampton
United Kingdom
[email protected]
Daniel Sohmen
Gene Center Munich, LMU Munich
Germany
[email protected]
Georg Stoecklin
German Cancer Research Center
Germany
[email protected]
Elizaveta Sokolova
Engelhardt Institute of Molecular Biology
Russian Academy of Sciences
Russian Federation
[email protected]
Mark Stoneley
MRC Toxicology Unit
United Kingdom
[email protected]
Joanna Somers
MRC Toxicology Unit
United Kingdom
[email protected]
Katja Sträßer
LMU Munich
Germany
[email protected]
Nahum Sonenberg
McGill University
Canada
[email protected]
Arunkumar Sundaram
Uni of Manchester
United Kingdom
[email protected]
c.uk
Poul Sorensen
BC Cancer Research Centre
Canada
[email protected]
Maxim Svetlov
Institute of Protein Research
Russian Federation
[email protected]
Christian Spahn
Charite - Universitätsmedizin Berlin
Germany
[email protected]
Naveen Swaroop
sri Venkateswara Veaterinary University
India
[email protected]
Nancy Standart
University of Cambridge
United Kingdom
[email protected]
Agata Swiatkowska
Institute of Bioorganic Chemistry Polish
Academy of Sciences
Poland
[email protected]
Page 294
List of Participants
Fraz Syed
Illumina
United States of America
[email protected]
T
U
Simon Uhse
University of Goettingen
Germany
[email protected]
Toma Tebaldi
CIBIO - University of Trento
Italy
[email protected]
Jernej Ule
UCL Institute of Neurology
United Kingdom
[email protected]
Ilya Terenin
Moscow State University
Russian Federation
[email protected]
Anna Urbanska
The International Institute of Molecular and Cell
Biology in Warsaw
Poland
[email protected]
Gabriel Therizols
Cancer Research Center of Lyon
France
[email protected]
Andreas Thess
CureVac GmbH
Germany
[email protected]
Klaske Thiadens
Sanquin Research
The Netherlands
[email protected]
Christopher Tiedje
Hannover Medical School
Germany
[email protected]
Bettina Tieg
Georg-August-University Göttingen
Germany
[email protected]
Peter Todd
University of Michigan
United States of America
[email protected]
Ivan Topisirovic
McGill University
Canada
[email protected]
V
Leos Valasek
Institute of Microbiology, AS CR
Czech Republic
[email protected]
Eivind Valen
Harvard University
United States of America
[email protected]
Petra Van Damme
University of Ghent
Belgium
[email protected]
Umesh Varshney
Indian Institute of Science
India
[email protected]
Veronica Venturi
Charles University
Czech Republic
[email protected]
John Verruto
Synthetic Genomics
United States of America
[email protected]
Page 295
EMBO Conference Series: Protein Synthesis and Translational Control
Silvia Verzini
University of Potsdam
Germany
[email protected]
Irena Vlatkovic
Max Planck Institute for Brain Research
Germany
[email protected]
Tobias von der Haar
University of Kent
United Kingdom
[email protected]
Klaus Wethmar
Max-Delbrueck-Center for Molecular Medicine
Germany
[email protected]
Hans-Joachim Wieden
University of Lethbridge
Canada
[email protected]
Anne Willis
Medical Research Council Toxicology Unit
United Kingdom
[email protected]
Marieke von Lindern
Sanquin Research and Landsteiner Lab.
AMC/UvA
The Netherlands
[email protected]
Daniel Wilson
University of Munich
Germany
[email protected]
Ottilie von Loeffelholz
EMBL Grenoble
France
[email protected]
Wolfgang Wintermeyer
MPI for Biophysical Chemistry
Germany
[email protected]
Vaclav Vopalensky
Charles University in Prague
Czech Republic
[email protected]
Noel Wortham
University of Southampton
United Kingdom
[email protected]
W
Susan Wagner
Academy of Science
Czech Republic
[email protected]
Elmar Wahle
University of Halle
Germany
[email protected]
David Weinberg
University of California, San Francisco
United States of America
[email protected]
Laurence Wurth
Center for Genomic Regulation (CRG)
Spain
[email protected]
X
Shifeng Xue
Stanford University
United States of America
[email protected]
Y
Akiko Yanagiya
McGill University
Canada
[email protected]
Page 296
List of Participants
Luke Yates
University of Oxford
United Kingdom
[email protected]
Eytan Zlotorynski
Nature Protocols
United Kingdom
[email protected]
Seda Yerlikaya
University of Geneva
Switzerland
[email protected]
Tao Zu
University of Florida
United States of America
[email protected]
Yael Yoffe
Weizmann Insistute of Science
Israel
[email protected]
Marat Yusupov
IGBMC
France
[email protected]
Gulnara Yusupova
IGBMC, CNRS
France
[email protected]
Itoh Yuzuru
Institut de Genetique et de Biologie Moleculaire
et Cellulaire (IGBMC)
France
[email protected]
Z
Lolita Zach
Technion
Israel
[email protected]
Angelica Raquel Zamudio Ochoa
Instituto de Fisiología Celular, UNAM
Mexico
[email protected]
Cleslei Zanelli
Univ Estadual Paulista – UNESP
Brazil
[email protected]
Elisabeth M Zielonka
EMBL Heidelberg
Germany
[email protected]
Page 297
EMBO Conference Series: Protein Synthesis and Translational Control
Page 298
Author's Index
The index number refers to the abstract number and not the page
number.
'
't Hoen, Peter A.C.
229
A
Abaeva, Irina
Adam, Julie
Adami, Valentina
Aeschimann, Florian
Aktinson, Gemma
Akulich, Kseniya
Alain, Tommy
Alard, Amadine
Albaret, Marie-Alexandra
Alexandrov, Kirill
Alkalaeva, Elena
Alleaume, Anne-Marie
Allwright, Mike
Altman, Roger
Altmann, Michael
Altmueller, Janine
Amadio, Marialaura
Anderson, Paul
Anderson, Ross
Andrade-Navarro, Miguel A.
Andreev, Dmitry
Andrews, Brenda J
Andreyanova, Ekaterina
Antic, Sanja
Antonarakis, Stylianos
Arava, Yoav
Archer, Stuart
Arenz, Stefan
Argentini, Manuela
Ariosa, Aileen
Arora, Smriti
Arpat, Bulak
Ashe, Mark
Astudillo, Patricio
Atkins, John
117
89
191, 211
66
19
47
238
166
39, 228
168
105, 123, 161, 217
85, 244
92
48
200
196
191
61
79
206, 235
47, 67, 214, 227
243
178
68
93
74
189
51, 69
76
42, 233
35
124
26, 84
148
22, 72, 197
EMBO Conference Series: Protein Synthesis and Translational Control
Avdulov, Svetlana
Averous, Julien
Aviner, Ranen
185
121
70
B
Bellato, Hermano Martins
Bianchi, Marco E.
Babic, Ana
Baggs, Eric
Bajer, Magdalena
Balmanno, Kathryn
Balsalobre, Aurelio
Balvay, Laurent
Ban, Nenad
Banez Coronel, Monica
Baranov, Pavel
Barbosa, Cristina
Barbosa-Silva, Adriano
Barna, Maria
Barriga de Vicente, Francisco
Bartel, David
Barth-Baus, Diane
Bar-Ziv, Lavi
Bash Imam, Zeina
Bastide, Amandine
Batlle, Eduard
Baudin-baillieu, Agnès
Bavli-Kertselli, Ira
Beadnell, Thomas
Becker, Christian
Beckmann, Benedikt
Beckmann, Roland
Behrmann, Elmar
Belin, Stéphane
Beljantseva, Jelena
Bell, Susanne
Belletir, Nicolette
Belova, Elena
Bennett, Jonathan
Berger, Audrey
Berger, Imre
Berninghausen, Otto
243
82
98
71
203
90
128
180, 181
2
64
72, 93, 160
53
235
59
148
6
159
74
228
73, 131, 215
148
170
74
185
196
120
41, 51, 69, 216
17
39, 86
135
22
24
178
114
123
42, 233
51, 69, 216
Author's Index
Bickel, Peter
Bienvienut, Willy
Biffo, Stefano
Biggin, Mark
Billas, Isabelle M. L.
Bitterman, Peter
Blanchard, Scott
Blanchet, Sandra
Blau, Christian
Bochkaeva, Zanda V.
Bock, Lars
Bock, Ralph
Boesch, Pierre
Bogdanov, Alexey
Bojarska, Elzbieta
Boland, Andreas
Boldrin, Paulo
Bolech, Michael
Bonaldi, Tiziana
Bornemann, Thomas
Bourdon, Jean-Christophe
Bousquet, Corinne
Bouvet, Philippe
Brackin, Robyn
Brandon, Harland
Braun, Thomas
Brauß, Thilo
Bremang, Michael
Brierley, Ian
Brina, Daniela
Bröcker, Markus
Brödel, Andreas
Bron, Patrick
Brook, Matthew
Broomhead, Helen
Brosch, Mario
Brüne, Bernhard
Brunelli, Silvia
Brünger, Katharina
Buderus, Victoria
Budkevich, Tanya
Bukau, Bernd
65
115
40, 75
65
34
185
43, 48
76
44
227
44
140
194
80, 178
77, 151
55
243
81
62
96, 119
39
138, 166
39
199
78
193
203
62
22
40
122
134
101, 115
79
133
104
203
82
222
203
17, 49
202
EMBO Conference Series: Protein Synthesis and Translational Control
Burakovskiy, Dmitry
Burgess, Hannah
Bushell, Martin
Buskirk, Allen
Buxbaum, Joseph
80
79
9, 73, 114, 131, 183, 215, 218, 221
18, 19
201
C
Celona, Barbara
Caceres, Carlos
Cain, Kelvin
Cairns, David
Calamita, Piera
Caldarola, Sara
Caliskan, Neva
Calkhoven, Cornelis
Camacho Villasana, Yolanda
Camara, Yolanda
Cambiaghi, Tavane
Cammas, Anne
Cannell, Ian G.
Carron, Coralie
Casola, Stefano
Castellano, Mar M
Castelli, Lydia
Castello, Alfredo
Castilho, Beatriz
Castillo, Estefania
Castro-Sanz, Ana B.
Cate, Jamie
Catez, Frederic
Cencic, Regina
Cenik, Elif Sarinay
Chambers, Jennifer M.
Charlet, Nicholas
Chattopadhyay, Samit
Chellini, Lidia
Chen, Wei
Chen, Ying
Chernova, Tatyana
Chew, Guo-Liang
Chommy, Hélène
Choudhury, K Roy
82
147
221
62
75
149
45
8
242
196
81
82, 162
9
181, 228
62
83
26
31, 85, 120, 186, 207, 244
81
148
83
48, 173
39, 86, 228
87
59
143
10
127
149
98
55
114
88
198
72
Author's Index
Christian, Helen
Ciesiolka, Jerzy
Cladiere, Lionel
Clancy, Jennifer
Clark, Susan
Clarke, Kim
Cleary, John
Cloonan, Nicole
Coatham, Mackenzie L.
Cobbold, Laura
Cockman, Matthew
Coldwell, Mark
Coleman, Mathew
Cong, Rong
Conrad, Dominik
Contreras, Vince
Cook, Simon
Cooke, Amy
Cope, Claire
Cornu, David
Corso, Andrew
Corthals, Garry
Costello, Joe
Cotobal, Cristina
Coureux, Pierre-Damien
Cowan, Joanne
Crechet, Jean-Bernard
Cridge, Andrew
Cui, Zhenling
Cunha, Carlos E.
Curk, Tomaz
Curran, Joseph
128
113, 225
158
187, 188
190
40
64
187
78
183
89
92, 182, 204, 220
23, 89, 100, 209
39
172
126, 165
90
91
90
76, 198
187
150
26, 84
27
158
92, 182, 204, 220
115
49
168
16
85
93
D
da Cunha, Carlos
D'Agostino, Vito
Darnell, Robert
Darzynkiewicz, Edward
Darzynkiewicz, Zbigniew
Das, Aritra
Das, Rhiju
Das, Saumitra
141
191
201
77, 133, 151
77
127
59
127
EMBO Conference Series: Protein Synthesis and Translational Control
Dassi, Erik
Davydov, Iakov
De Bo, Christof
de Klerk, Eleonora
de Ru, Arnoud
Décimo, Didier
Defenouillère, Quentin
Demeshkina, Natalia
Denecke, Bernd
Derry, Brent
des Georges, Amedee
Deslongchamps, Pierre
Desnoyers, Guillaume
Dever, Thomas
Dhote, Vidya
Diaz, Jean-Jacques
Dikstein, Rivka
DiMarco, Sergio
Dinman, Jonathan
Dmitriev, Sergey
Donato, Rosario
Dondapati, Srujan
Dong, Jinsheng
Dontsova, Olga
D'Orchymont, Arnaud
Dormoy-Raclet, Virginie
Dorner, Silke
Doudna, Jennifer
Drapeau, Elodie
Draycheva, Albena
Drouin, Jacques
Dubrac, Alexandre
Duncan, Caia
Duncan, John
Dunn, Joshua
Dutkiewicz, Mariola
Duval, Mélodie
Dworkin, Jonathan
Dyer, Nigel
94, 226
44
160
229
22
180, 181
54
15
142
21
37
87
95
18
37
39, 86, 228
5
82, 107
43
47, 67, 214, 227
82
134
167
80, 178
38
82
68
173
201
96
128
162
27
102
24
113
97
20
11
E
Easton, Andrew
11
Author's Index
Eaton, Lucinda
Eckmann, Christian
Edwards, Richard
Ehrl, Benno
Eichelbaum, Katrin
Eichwald, Sabina
Eiler, Daniel
Eillis, Anne E.
Eliseev, Boris
El-Khawand, Sally
Elroy-Stein, Orna
Emara, Mohamed
Eperon, Ian C
Epstein, Irina
Erfurth, Corinna
Eriani, Gilbert
Eskelin, Katri
Evfratov, Sergey
182
174
92
222
31, 120
8
34
9
105, 217
166
70
61
184
98
172
38
99
178
F
Fuchs, Helmut
Fabbretti, Attilio
Falciani, Francesco
Fang, Ying
Fechter, Pierre
Fehr, Carmen
Feng, Tianshu
Fernandez, Javier
Fernández-Bautista, Nuria
Ferreira, Celine
Ficner, Ralf
Fieulaine, Sonia
Firczuk, Helena
Firth, Andrew
Fischer, Bernd
Fischer, Jeffrey J.
Fischer, Niels
Fischer, Roman
Fleurdépine, Sophie
Foehr, Sophia
Föhr, Sophia
Fok, Patrick
40
34, 97
40
22
97
172
23, 100
157
83
9
136, 144
101, 115
102
22, 72
31, 85, 120, 186, 244
78
44, 50
23, 89
56
85, 120, 186, 244
31
111
EMBO Conference Series: Protein Synthesis and Translational Control
Formenti, Fabio
Fox, Gearoid
Fox, Paul
Francisco, Rosario
Frank, Joachim
Frank, Peske
Fraser, Christopher
Frias, Maria
Friday, Andrew
Fritsch, Claudia
Frolova, Ludmila
Fromont-Racine, Micheline
Frost, Sigal
Fuchsbauer, Olivier
89
160
32
157
37
141
195
201
126, 165
104
105
54
106
97
G
Gaestel, Matthias
Gailus-Durner, Valérie
Galicia Vazquez, Gabriela
Gallo, Simone
Gallouzi, Imed
Gallouzi, Imed-Eddine
Galvão, Fabio Carrilho
Gamm, Magdalena
Gammage, Payam
García Guerrero, Aldo
García, María
García-Beyaert, Marina
Garreau de Loubresse, Nicolas
Gatfield, David
Gautheret, Daniel
Gavis, Elizabeth
Gawron, Daria
Ge, Wei
Gebauer, Fátima
Geiger, Tamar
Geng, Songsong
Gevaert, Kris
Ghayad, Sandra
Giering, Florian
Giese, Anne
Giglione, Carmela
191
40
87, 107
75
107
82
243
108
194
242
28
109
46, 110
124
170
24
7
89, 100, 209
28, 109
70
111
7
39, 86
172
140
101, 115
Author's Index
Gilbert, Robert
Gilbert, Robert J. C.
Gilley, Rebecca
Gillis, Laura
Giraldez, Antonio
Girodat, Dylan
Gismondi, Angelo
Glatt, Sebastian
Godiyenko, Yuliya
Gomez-Garcia, Jose
Gorgoni, Barbara
Gorska, Agnieszka
Gould, Phillip
Graham, Robert
Granatino, Nicolas
Grant, Chris
Grassucci, Robert A.
Gray, Nicola
Green, Rachel
Grewal, Savraj
Grieve, Kelsey
Grill, Jacques
Grishin, Alexander
Grosso, Stefano
Grubmuller, Helmut
Grundy, Richard G.
Grzela, Renata
Gualerzi, Claudio O.
Guichard, Sylvie
Guimarães, Beatriz
Guitton, Jérôme
Gulay, Suna
Gutierrez, Erik
23, 56
198
90
95
58
236
149
112
237
185
33
113, 225
11
188
100
26, 84, 223
37
79
192
169
33
9
105
114
44
9
101, 115
34
90
101
228
43
18
H
Habeck, Michael
Habermann, Bianca
Hackmann, Alexandra
Hacot, Sabine
Haimov, Ora
Haller, Andrea
Hammond, Gifty
216
196
230
39, 86, 228
5
97
122
EMBO Conference Series: Protein Synthesis and Translational Control
Hampe, Jochen
Haneke, Katharina
Hanson, Johannes
Hartleben, Götz
Hashem, Yaser
Hatin, Isabelle
Hauryliuk, Vasili
Hazemann, Isabelle
He, Fang
Hellen, Christopher
Henderson, Melissa
Hendriks, Gert-Jan
Hentze, Matthias
Herbert, Hayley
Herrera, Jeremy
Herrmann, Alexander
Hershey, John
Heus, Hans
Heymann, Felix
Hezwani, Meowea
Hietakangas, Ville
Hildebrand, Peter
Hinnebusch, Alan
Hodges, Samantha
Hoffmeister, Christian
Holtkamp, Wolf
Horenk, Juliane
Horos, Rastislav
Horvilleur, Emilie
Hrabê de Angelis, Martin
Huang, David CS
Huard, Karine
Hubbard, Simon
Huidobro-Toro, Juan Pablo
Huikuri, Heikki
Humphreys, David
Huntzinger, Eric
Huse, Klaus
Hynes, Carly
104
116
108, 139
8
37
170
135
34
10
37, 117
103, 126
66
31, 85, 91, 120, 186, 207, 244
92
185
104
195
118
142
92
145
17
36
182
134
16, 119
222
31, 120, 164, 186
9
40
143
233
26, 84
147
125
188, 190
55
104
188
I
Iadevaia, Valentina
121, 149
Author's Index
Ingolia, Nicholas
Irie, Kenji
Ismer, Jochen
Itoh, Yuzuru
Ivanov, Ivaylo
Ivanov, Pavel
Ivanova, Elena
Izaurralde, Elisa
13, 72, 212
130
17
122
197
61
123
55
J
Jabnoune, Mehdi
Jacquier, Alain
Jakson, Thomas
Jan, Eric
Jang, Sung Key
Janich, Peggy
Jankowska-Anyszka, Marzena
Jaquier-Gubler, Pascale
Jaworski, Jacek
Jemielity, Jacek
Jenner, Lasse
Jennings, Martin
Jochmann, Viviane
Johnston, Harvey
Johnston, Samantha
Jonas, Stefanie
Jonathan, Weissman
Jones, Kevin
Jones, Russel G
Jordan, Lee
Jukes-Jones, Rebekah
Juli, Giada
60
54
73
21
30
124
77
93
231
151
15
4
49
182
215
55
24
182
21
39
221
149
K
Kaija, Helena
Kalaora, Rinat
Kamenska, Anastasiia
Kanke, Matt
Karuppasamy, Manikandan
Karyagina, Anna
Katoch, Aanchal
Keiper, Brett
125
240
133, 218
152
42, 233
105
127
103, 126, 165
EMBO Conference Series: Protein Synthesis and Translational Control
ndent translati26, 84
Kessler, Benedikt
Khan, Debjit
Khetchoumian, Konstantin
Khutornenko, Anastasia A.
Kiebler, Michael
Kilday, John-Paul
Kim, Goheun
Kim, Joo-Ran
Kimchi, Adi
Kimura, Yuichi
Kirmizialtin, Serdal
Klaholz, Bruno
Klingenspor, Martin
Knight, John
Knight, John R.P.
Knoops, Kèvin
Kolb, Vyacheslav
Kondrashov, Alexander
Konevega, Andrey L.
Konieczny, Steven
Korepanov, Alexey
Kortelainen, Marja-Leena
Kostoska, Galena
Kotini, Suresh
Kousar, Rehana
Kowalska, Joanna
Kramer, Günter
Krans, Amy
Krawczak, Michael
Krebber, Heike
Krijgsveld, Jeroen
Krüger, Marcus
Kryuchkova, Polina
Kubacka, Dorota
Kubick, Stefan
Kuchly, Claire
Kudrin, Pavel
Kuersten, Scott
Kuhle, Bernhard
Kunze, Michael
Kuzuoglu-Öztürk, Duygu
26, 84
23, 89, 100
127
128
214
129
9
152
18
240
130
43
34, 38, 97, 158
40
131
73
42, 233
224
9
16
138
97
125
226
132
163
151
202
10
104
230
31, 85, 120, 186, 244
193
105
133, 218
134
170
135
29
136, 144
203
55
Author's Index
L
Lacerda, Rafaela
Laforêts, Florian
Lai Kee Him, Joséphine
Lal, Ridhima
Landry, Dori M.
Langlois, Robert
Larquet, Eric
Larsson, Nils-Göran
Lasfargues, Charline
Lastdrager, Jeroen
Lecampion, Cécile
Lee, Jong–Bong Lee
Lee, Sejeong
Lee, Seung Hwan
Lee, Su Jung
Legen, Julia
Legendre, Rachel
Leger, Melissa
Legrand, Pierre
Lehweß-Litzmann, Anja
Leicht, Stefan
Leidel, Sebastian
Leprivier, Gabriel
Leutz, Achim
Lewis, Stephen M.
Li, Gene-Wei
Li, Jingyi Jessica
Li, Yanhua
Lian, Xian Jin
Liao, Yalin
Lieberman, Judy
Liepelt, Anke
Limousin, Taran
Lindqvist, Lisa
Lisi, Gaia
Liu, Rui
Liu, Yi
Liu, Ying
Liu, Yuanjing
Liu-Yi, Phebee
137, 156
228
101
127
147
37
158
196
138, 166
139
60
30
119
30
49
140
170
210
101
141
186
171, 208
21
206, 235
95
72
65
22
82
186
61
142
176
143
149
121
144
145
64
89
Page 311
EMBO Conference Series: Protein Synthesis and Translational Control
Livneh, Etta
Loerke, Justus
Loewith, Robbie
Loguercio Polosa, Paola
Lomakin, Ivan
Lopez-Lastra, Marcelo
Loreni, Fabrizio
Lorsch, Jon
Lu, Wei-ting
Lucas, Christian M
Lührmann, Reinhard
Lukash, Tetyana
Lukaszewicz, Maciej
106
17
239
196
47, 146
147, 148
149
167
218
184
57
150
77
M
Macdonald, Paul
MacFarlane, Marion
Mackeen, Mukaram
Mah, Nancy
Mäkinen, Kristiina
Malina, Abba
Mallucci, Giovanna
Malys, Naglis
Mancera-Martinez, Eder
Mancino, Marilena
Mancuso, Francesco
Manivel, J. Carlos
Mann, Matthias
Marcel, Virginie
Mark, Petersen
Marques-Ramos, Ana
Martin, Franck
Martineau, Yvan
Martinez, Magdalena
Martinez-Salas, Encarna
Marx, Gernot
Marzi, Stefano
Mata, Juan
McArthur, Kate
McCarthy, John
McGeachy, Anna
Mechulam, Yves
152
114
89
206
99
87
73, 131, 153
154
155
75
62
185
222
39, 228
185
137, 156
38
138, 166
237
157
142
34, 97
27
143
102, 154
212
158
Author's Index
Meharg, Caroline
Meinnel, Thierry
Meister, Gunter
Melamed, Daniel
Menetret, Jean-François
Meng, Xiang
Mengardi, Chloe
Menschaert, Gerben
Mercier, Evan
Merrick, William
Mertani, Hichem C
Mestdagh, Claire
Metodiev, Metodi
Meusburger, Madeleine
Mhlanga, Musa
Michel, Audrey
Micura, Ronald
Mielke, Thorsten
Mihailovic, Marija
Mikhaylova, Tatiana
Millevoi, Stefania
Mills, Eric
Milón, Pohl
Miluzio, Annarita
Minczuk, Michal
Minshall, Nicola
Mironov, Andrey
Mo, Fan
Modelska, Angelika
Mohammad-Qureshi, Sarah
Moore, Kat
Morel, Anne-Pierre
Morel, Benjamin
Morrison, J. Kaitlin
Mossanen, Jana C.
Mouaikel, John
Mozaffari-Jovin, Sina
Mrusek, Devid
Mühlemann, Oliver
Müller, Christine
Müller, Christoph W
Müller, David
196
101, 115
172
74
38, 158
102
176
7
236
159
39, 228
170
196
239
199
72, 160
97
17
62
161
162
13
80
40
194
133, 218
47
236
94
163
164
39
162
103, 126, 165
142
54
57
134
52
8
112
138, 166
EMBO Conference Series: Protein Synthesis and Translational Control
Munoz, Antonio
Mureev, Sergey
Muro, Enrique M.
Murphy, Fiona
Myasnikov, Alexander G.
167
168
206
114
34
N
Nagarajan, Sabarish
Nagy, Andras
Nakas, Apostolos
Nalbantoglu, Josephine
Namane, Abdelkader
Namy, Olivier
Nandi, Ipsita
Napthine, Sawsan
Navarrete, Camilo
Nedialkova, Danny
Negrutskii, Boris
Nelissen, Frank
Neschen, Susanne
Neumann, Angelina
Niepmann, Michael
Nieradka, Andrzej
Nierhaus, Knud
Nikolaev, Sergey
Noeske, Jonas
Norbury, Chris
Nothnagel, Michael
Nousch, Marco
Nusbaum, Julien
169
187
114
111
54
76, 170, 198
63
22
147
171
150
118
40
172
172
229
17
93
173
56
104
174
101, 115
O
O'Brien, Edward
O'Connor, Patrick BF
O'Day, Elizabeth
Offenhauser, Nina
Oh, Jungsic
Oh, Seok Yoon
Ohlmann, Theo
Ohlmann, Théophile
O'Kelly, Ita
Olivares, Eduardo
175
72, 93, 160
61
40
30
10
176
180, 181
204
147
Author's Index
Oliveto, Stefania
Olsthoorn, René
Ostareck, Dirk H.
Ostareck-Lederer, Antje
Osterman, Ilya
Ott, Sascha
75
177
142
142
178
11
P
Paek, Ki Young
Papasaikas, Panagiotis
Paleskava, Alena A.
Panda, Amaresh
Pandey, Poonam
Panthu, Baptiste
Park, Chan Bae
Park, Jonghyun
Parker, Brian
Parker, Matthew
Paron, Igor
Patel, Hardip
Pauli, Andrea
Pavitt, Graham
Peil, Lauri
Peixeiro, Isabel
Pelletier, Jerry
Pereira, Catia
Pereira, Sandro
Peretti, Diego
Pérez Martínez, Xochitl
Perry, Lisa
Pesce, Elisa
Peske, Frank
Pestova, Tatyana
Pfister, Stefan
Philipp, Janine
Pichon, Xavier
Piecyk, Karolina
Pierre, Philippe
Piñeiro, David
Pino, Karla
Pitkänen, Leena
Platzer, Matthias
30
28
50
179
179
180, 181
196
30
190
185
222
187, 188, 190
88
4, 26, 84, 163
19
53
87, 107
81
20
73, 131
242
182
75
16, 45, 132
37, 117
21
205
73, 183
77
87
157, 184
147
99
104
EMBO Conference Series: Protein Synthesis and Translational Control
Poirier, Yves
Pollak, Michael
Pollard, Patrick
Poller, Jonathan
Polunovsky, Vitaly
Poria, Dipak
Porvari, Katja
Pospisek, Martin
Pöyry, Tuija
Pozza, Alexandre
Prats, Anne-Catherine
Prats, Hervé
Preiss, Thomas
Prokhorova, Irina
Proud, Christopher
Provenzani, Alessandro
Puget, Stephanie
Puglisi, Joseph D.
Puisieux, Alain
Pulk, Arto
Puri, Mira
Pyronnet, Stéphane
60
21
89
106
185
63
125
234
9, 184
101, 115
39
162
31, 186, 187, 188, 189, 190
46, 80, 110
21, 121, 149, 220, 237
191
9
34
39
48
187
138, 166
Q
Quast, Robert
Quattrone, Alessandro
134
94, 211, 226
R
Radhakrishnan, Aditya
Raisch, Tobias
Rajman, Marek
Ramachandran, Vinoy
Ramajo, Jorge
Ramrath, David
Ranum, Laura
Ratcliffe, Peter
Rathkolb, Birgit
Raveh-Amit, Hadas
Ravindar, Kontham
Ray, Partho Sarothi
Regev, Aviv
Reid, Tammy
192
55
193
154
157
17
64
23, 89, 100
40
106
87
63
88
64
Author's Index
Rejman, Dominik
Remke, Marc
Remme, Jaanus
Rho, Sangchul
Rhoads, Robert
Ricci, Emiliano
Ricciardi, Sara
Richardson, William
Rinn, John
Riuzzi, Francesca
Rizzacasa, Mark A.
Robaglia, Christophe
Robert, Francis
Robinson, Carol
Roblin, Pierre
Robool, Joanne
Rodnina, Marina
Romão, Luísa
Romby, Pascale
Roobol, Jo
Roobol, Joanne
Rorbach, Joanna
Rosa-Calatrava, Manuel
Rossi, Danuza
Rossi, Federico
Rowe, William
Rozman, Jan
Rozov, Alexey
Rubilar, Paulina
Rübsamen, Daniela
Rubtsova, Maria
Ruzzenente, Benedetta
Ryabova, Lyubov
Ryu, Young Hee
135
21
19
30
126
176
40
33, 79
88
82
143
60
87
237
34
73
16, 44, 45, 50, 80, 132, 141
53, 137, 156
97
131
73, 131
194
181
195, 243
147
26, 84
40
15
176
203
178
196
12, 155
152
S
Sachs, Matthew
Sachse, Rita
Sagar, Vinay
Saguy, Matthieu
Saini, Preeti
Samhita, Laasya
197
134
149
198
18
35
EMBO Conference Series: Protein Synthesis and Translational Control
Sanbonmatsu, Karissa
Sanderson, Rebecca
Santos, Karine
Särkioja, Terttu
Sattlegger, Evelyn
Saurin, Jean-Christophe
Savage, G. Paul
Saveanu, Cosmin
Savulescu, Anca
Sawicka, Kirsty
Saxena, Manisha
Sbarrato, Thomas
Schaeffer, Laure
Schäfer, Heiner
Schaffitzel, Christiane
Scharff, Lars
Scheckel, Claudia
Scheerer, Patrick
Scherrer, Anne
Schibich, Daniela
Schier, Alexander
Schmid, Tobias
Schmidt, Andrea
Schmitt, Emmanuelle
Schmitz-Linneweber, Christian
Schneider, Robert J
Schoehn, Guy
Schofield, Christopher J.
Schofield, James
Schott, Johanna
Schratt, Gerhard
Schreiber, Stefan
Schröder, Gunnar F.
Schulz, Julia
Schuman, Erin
Schumann, Frank
Schuster, Birgit
Schuurmans, Jolanda
Schwarz, Juliane
Schwertfeger, Kaylee
Seberg, Andrew
Secco, David
17, 43
163
57
125
49, 81
39, 228
143
54
199
183
200
114
38
205
42, 233
140
201
17
123
202
88
203
17
158
140
166
42, 233
89, 100, 209
204
205
193
104
44, 50
206
98
104
207
139
208
185
49
60
Author's Index
Sekine, Shun-ichi
Sekirnik, Rok
Sellier, Chantal
Semler, Bert
Sergeeva, Olga
Sergiev, Petr
Shan, Shu-ou
Shanmugam, Renuka
Shapira, Michal
Sharathchandra, Arandkar
Shatsky, Ivan
Shchepetilnikov, Mikhail
Shetty, Sunil
Shin, Byung-Sik
Shu, Qingyao
Shung, Chang
Sibbritt, Tennille
Sidarovich, Viktoryia
Sidrauski, Carmela
Siebert, C. Alistair
Simonetti, Angelita
Simpson, Clare
Sims, Paul
Sinfield, Oliver
Singh, Pooja
Singleton, Rachelle
Sinitsyn, Pavel
Sinvani, Hadar
Skucha, Anna
Smales, Mark
Smirnova, Victoria
Smith, Ewan
Smith, Joel
Smith, Karen
Smith, Nicola
Smith, Paul
Smith, Richard
Snijder, Eric
Soengas, Marisol
Sohmen, Daniel
Sokolova, Elizaveta
Solano-Gonzàlez, Eduardo
122
100, 209
10
71
80
80, 178
42, 233
81
210
127
47, 67, 214, 227
12
35
18
60
17
190
211
212
198
34, 38
213
26, 84
102
23
89
47
5
68
73, 131
47
215
33
185
188
90
33
22
28
216
217
39
EMBO Conference Series: Protein Synthesis and Translational Control
Solis, Loretto
Söll, Dieter
Somaiah, Ragan
Somers, Joanna
Sonenberg, Nahum
Song, Anren
Sorci, Guglielmo
Sorensen, Poul H
Soto Rifo, Ricardo
Sousa Martins, Joao
Spahn, Christian
Spåhr, Henrik
Spriggs, Keith
Spriggs, Ruth
Springer, Mathias
Squires, Jeffrey
Standart, Nancy
Stanhill, Ariel
Stark, Holger
Starosta, Agata
Stech, Marlitt
Steen, Judith
Steinmetz, Lars
Steitz, Thomas
Stelmaszuk, Natalia
Stepinski, Janusz
Stewart, Joanna
Stoecklin, Georg
Stoneley, Mark
Strasser, Andreas
Sträßer, Katja
Strein, Claudia
Strub, Katharina
Subash, Jacob
Suetsugu, Shiro
Sun, Xiao-Ming
Sun, Zhi
Sundaram, Arunkumar
Svetlov, Maxim
Svitkin, Yuri
Swanson, Mark
Swiatkowska, Agata
148
122
87
9
1, 5, 238
126
82
21
176
79
43, 49
196
183
183
97
190
133, 213, 218
219
44, 50
19
134
86
31
34, 146
151
77
220
116, 205
183, 221
143
222
31
123
126
122
114
22
223
224
5
49
113, 225
Author's Index
Szafranski, Karol
104
T
Tacke, Frank
Tafer, Yasmine
Tanaka, Junichi
Tas, Ali
Taylor, Michael
Taylor, Peter
Tebaldi, Toma
Teixeira, Alexandre
Tenson, Tanel
Terenin, Ilya
Teulade-Fichou, Marie-Paule
Therizols, Gabriel
Thérizols, Gabriel
Thiadens, Klaske A.M.H.
Thompson, Alistair
Thompson, Andy
Thompson, Sunnie R.
Tian, Siqi
Tiedje, Christopher
Tieg, Bettina
Tnimov, Zakir
Todd, Peter
Tonge, Pete
Touriol, Christian
Treffers, Emmely
Troncoso, Juan
Tsai, Albert
Tuomainen, Päivi
Tuschev, Georgi
Tzach, Lolita
142
39
87
22
21
121
226
53, 137, 156
19, 135
47, 67, 214, 227
177
228
39
229
39
101
147
59
191
230
168
10
187
162
22
64
34
99
98
219
U
Uhse, Simon
Ule, Jernej
Ungewickell, Charlotte
Urbanska, Anna
230
25
216
231
V
Vaiana, Andrea
34, 44
EMBO Conference Series: Protein Synthesis and Translational Control
Valen, Eivind
Valentini, Sandro Roberto
Van Criekinge, Wim
Van Damme, Petra
van der Giessen, Kate
van der Meer, Yvonne
van Veelen, Peter
van Vugt-Jonker, Aafke
Varano, Gabriele
Varik, Vallo
Varshney, Umesh
Vendrell, Julie
Viero, Gabriella
Vikström, Ingela
Virumäe, Kai
Vitale, Elena
Vlasov, Peter
Vlatkovic, Irena
von der Haar, Tobias
von Lindern, Marieke
von Loeffelholz, Ottilie
Vopalensky, Vaclav
88
195, 243
7
7
82
22
22
118
62
135
35
39
226
143
19
62
217
98
232
164, 229
42, 233
234
W
Wagner, Gerhard
Wahl, Markus
Walter, Peter
Wang, Leyi
Wappner, Pablo
Warren, Peter
Wasserman, Michael
Webb, Andrew
Weber, Gert
Wei, Jiajie
Weichenrieder, Oliver
Weinberg, David
Weissman, Jonathan S
Wethmar, Klaus
Wieden, Hans-Joachim
Wilhelm, Brian
Willis, Anne
Wilson, Daniel
61, 210
57
212
48
89
86
48
143
57
197
55
6
72
206, 235
78, 236
82
73, 114, 131, 183, 184, 215, 221
19, 51, 69, 216
Author's Index
Wilson, Daniel N.
Wilson, Lindsay A.
Wintermeyer, Wolfgang
Wohlbold, Lara
Wolf, Alex
Wolf, Eckhard
Woolstenhulme, Christopher
Wortham, Noel
Wurth, Laurence
19, 51, 69, 216
9
16, 96, 119, 141
55
209
40
18, 19
237
28
X
Xue, Shifeng
59
Y
Yamamoto, Atsushi
Yanagiya, Akiko
Yang, Ming
Yángüez, Emilio
Yates, Luke
Yee, Douglas
Yerlikaya, Seda
Yoffe, Yael
Yokoyama, Shigeyuki
Yu, Chien-Hung
Yusupov, Marat
Yusupova, Gulnara
23, 89, 100
238
100
83
56
185
239
240
122
177
15, 43, 46, 110
15, 46, 110
Z
Zach, Lolita
Zamudio Ochoa, Angelica Raquel
Zanelli, Cleslei
Zayer, Adam
Zhang, Xin
Zhang, Ze
Zhe, Wang
Zhou, Yu
Zidek, Laura
Zielonka, Elisabeth M
Zinoviev, Alexandra
Zivojnovic, Marija
Zu, Tao
Zytek, Malgorzata
241
242
195, 243
100, 209
42, 233
121
50
4
8
244
210
82
64
151
EMBO Conference Series: Protein Synthesis and Translational Control
Page 324
Notes
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EMBO Conference Series: Protein Synthesis and Translational Control
Page 326
Notes
Page 327
EMBO Conference Series: Protein Synthesis and Translational Control
Page 328
Notes
Page 329
EMBO Conference Series: Protein Synthesis and Translational Control
Page 330
Notes
Page 331
EMBO Conference Series: Protein Synthesis and Translational Control
Page 332
Notes
Page 333
EMBO Conference Series: Protein Synthesis and Translational Control
Page 334