UMEÅ UNIVERSITY DISSERTATIONS
ISBN 978-91-7601-355-7
Functional Aspects of Modified Nucleosides in tRNA
Hao Xu
Department of Molecular Biology
Umeå University
Umeå, Sweden, 2015
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Copyright © 2015 Hao Xu
ISBN: 978-91-7601-355-7
Printed by: Print & Media
Umeå, Sweden, 2015
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To my parents for everything
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Table of Contents
Appended papers ...................................................................................................... 2
Abbreviations ........................................................................................................... 3
Abstract .................................................................................................................... 4
1. Introduction .......................................................................................................... 5
1.1 Transfer RNA ................................................................................................. 5
1.2 Modified nucleosides in tRNA ....................................................................... 6
1.2.1 Wobble uridine modifications and modifying enzymes .......................... 6
1.2.2 tRNA modifying enzyme required for m1A58 formation.......................... 8
1.2.3 tRNA surveillance ................................................................................... 8
1.3 Translation ...................................................................................................... 9
1.3.1 The translation process ............................................................................ 9
1.3.2 Translation errors and frameshifting ..................................................... 10
1.3.3 Role of wobble uridine modifications in translation ............................. 12
1.4 Elongator complex........................................................................................ 13
1.4.1 Elongator complex in yeast Saccharomyces cerevisiae ........................ 13
1.4.2 Regulation of Elongator complex activity ............................................. 14
1.5 The PKC family ............................................................................................ 15
Results and discussion ............................................................................................ 17
Conclusions ............................................................................................................ 22
Acknowledgements ................................................................................................ 23
References .............................................................................................................. 25
Paper I-III
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Appended papers
This thesis is based on the following appended papers (reproduced with permission
from the publishers), which are referred to by the corresponding Roman numerals
in the text.
I.
The role of wobble uridine modifications in +1 translational frameshifting
in eukaryotes.
H Tükenmez*, H Xu*, A Esberg and AS Byström (2015)
Nucleic Acids Research. 17 Aug 2015. doi: 10.1093/nar/gkv832
*These authors contributed equally
II.
Yeast Elongator protein Elp1p does not undergo proteolytic processing in
exponentially growing cells.
H Xu, J Bygdell, G Wingsle and AS Byström (2015)
MicrobiologyOpen. 30 Jul 2015. doi: 10.1002/mbo3.285
III.
TRM6/61 connects PKCα with translational control through tRNA iMet
stabilization: impact on tumorigenesis.
F Macari, Y El-houfi, G Boldina, H Xu, S Khoury-Hanna, J Ollier,
L Yazdani, G Zheng, I Bièche, N Legrand, D Paulet, S Durrieu,
AS Byström, S Delbecq, B Lapeyre, L Bauchet, J Pannequin, F Hollande,
T Pan, M Teichmann, S Vagner, A David, A Choquet and D Joubert
Oncogene. 3 Aug 2015. doi: 10.1038/onc.2015.244
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Abbreviations
Acetyl-coA
Acetyl coenzyme A
ASL
Anticodon Stem Loop
cm5U
5-carboxymethyluridine
eIF
Eukaryotic Initiation Factor
eEF
Eukaryotic Elongation Factor
eRF
Eukaryotic Release Factor
GBM
Glioblastoma Multiforme
HAT
Histone Acetylation Transferase
IRES
Internal Ribosome Entry Site
LC-MS
Liquid Chromatography-Mass Spectrometry
m1A
1-methyladenosine
mcm5U
5-methoxycarbonylmethyluridine
mcm5s2U
5-methoxycarbonylmethyl-2-thiouridine
MTases
Methyltransferases
ncm5U
5-carbamoylmethyluridine
ncm5Um
5-carbamoylmethyl-2’-O-methyluridine
NLS
Nuclear Localization Signal
NMR
Nuclear Magnetic Resonance
PKC
Protein Kinase C
Pol II
RNA Polymerase II
Pol III
RNA Polymerase III
SAM
S-adenosyl Methionine
TAP
Tandem Affinity Purification
TCA
Trichloroacetic Acid
TM Tag
Tandem Mass Tag
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Abstract
Transfer ribonucleic acids (tRNAs) are extensively modified, especially their
anticodon loops. Modifications at position 34 (wobble base) and 37 in these loops
affect the tRNAs’ decoding ability, while modifications outside the anticodon
loops, e.g. m1A58 of tRNA iMet , may be crucial for tRNA structure or stability. A
number of gene products are required for the formation of modified nucleosides,
e.g. at least 26 proteins (including Elongator complex) are needed for U34
modifications in yeast, and methyl transferase activity of the Trm6/61p complex is
needed to form m1A58. The aim of the studies which this thesis is based upon was to
investigate the functional aspects of tRNA modifications and regulation of the
modifying enzymes’ activity.
First, the hypothesis that ncm5U34, mcm5U34, or mcm5s2U34 modifications may
be essential for reading frame maintenance was investigated. The results show that
mcm5 and s2 group of mcm5s2U play a vital role in reading frame maintenance.
Subsequent experiments showed that the +1 frameshifting event at Lys AAA codon
occurs via peptidyl-tRNA slippage due to a slow entry of the hypomodified tRNALys.
Moreover, the hypothesis that Elp1p N-terminal truncation may regulate
Elongator activity was investigated. Cleavage of Elp1p was found to occur between
residue 203 (Lys) and 204 (Ala) and to depend on the vacuolar protease Prb1p.
However, including trichloroacetic acid (TCA) during protein extraction abolished
the appearance of truncated Elp1p, showing that its truncation is a preparation
artifact.
Finally, in glioma cell line C6, PKCα was found to interact with TRM61. RNA
silencing of TRM6/61 causes a growth defect that can be partially suppressed by
tRNA iMet overexpression. PKCα overexpression reduces the nuclear level of TRM61,
likely resulting in reduced level of TRM6/61 complex in the nucleus. Furthermore,
lower expression of PKCα in the highly aggressive GBM (relative to its expression
in less aggressive Grade II/III glioblastomas) is accompanied by increased
expression of TRM6/61 mRNAs and tRNA iMet , highlighting the clinical relevance of
the studies.
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1. Introduction
1.1 Transfer RNA
In Eukaryotes, transfer ribonucleic acid (tRNA) precursors are generated by
transcription of tRNA genes, mediated by Pol III (RNA polymerase III), then
mature tRNA species are produced via the actions of various endo- and exonucleases and tRNA modifying enzymes (Phizicky & Hopper, 2010). Mature
tRNAs are typically 75 to 90 nucleotides long and have a cloverleaf-like
secondary structure with: an acceptor stem, generated by the 5’ end and 3’ CCA
end of the tRNA; a D stem and loop, containing dihydrouridine (D); an anticodon
stem and loop, which has a triplet-nucleotide anticodon at position 34-36; a
variable loop; and a TΨC stem and loop, containing ribothymidine (T),
pseudouridine (Ψ) and cytosine (C) at position 54 to 56 in almost all tRNAs (Fig.
1). Due to base stacking and hydrogen bonding between the D and TΨC loops the
cloverleaf-like secondary structure is twisted into an L-shaped tertiary structure,
leaving the acceptor stem with the 3’ (CCA) end and the anticodon loop at two
distal extremities of the tRNA (Kim et al., 1974) (Fig.1). The amino acid carried by
the tRNA is covalently bond to the 3’-hydroxyl group on the CCA end with the aid
of aminoacyl tRNA synthetases (Ibba & Soll, 2000).
Figure 1. Secondary structure (left) and tertiary structure (Kim et al., 1974)
(right) of tRNA.
The yeast Saccharomyces cerevisiae produces 42 cytoplasmic tRNA species that
decode the 61 sense codons (Percudani et al., 1997, Hani & Feldmann, 1998). Thus,
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some tRNAs are capable of reading more than one codon, a feature explained by
the wobble hypothesis (Crick, 1966). According to this hypothesis, canonical
Watson-Crick base pairing (A with U and C with G) occurs between the first two
nucleotides of the codon and the nucleotides at position 36 and 35 in the anticodon
of a tRNA. However, the nucleotide at position 34 can also pair with the third base
of the codon through non-canonical base pairing. Therefore, position 34 of a tRNA
is called the wobble position.
1.2 Modified nucleosides in tRNA
Transfer RNA molecules are post-transcriptionally modified via processes that
generate derivatives of adenosine (A), guanosine (G), uridine (U) and cytidine (C)
at various positions. To date, more than 100 modified nucleosides have been
identified in tRNAs from Archaea, Bacteria, and Eukarya (Machnicka et al., 2013).
These modified nucleosides are distributed all over tRNAs. Among all the
modified nucleosides observed in tRNA, eight are present at the same position and
the same subpopulation of tRNA isoacceptors in all domains of life, suggesting that
tRNA modifications have highly conserved origins and functions (Björk, 1986,
Björk et al., 2001). Modified nucleosides in the anticodon loop, especially
positions 34 (the wobble position) and 37, often affect tRNA’s decoding abilities
by restriction or improvement of codon-anticodon interactions, which may affect
reading frame maintenance (Agris, 1991, Björk, 1995, Johansson et al., 2008, Lim,
1994, Yokoyama & Nishimura, 1995). Modified nucleosides present in the
anticodon loop may also be required for aminoacylation (Giege et al., 1998), while
some modifications outside the anticodon loop, e.g. m1A58 of tRNA iMet , are vital for
the structure or stability of the tRNA (Anderson et al., 1998, Calvo et al., 1999,
Kadaba et al., 2004). Hereafter, the thesis focuses mainly on wobble uridine (U34)
modifications and A58 methylation of tRNA iMet .
1.2.1 Wobble uridine modifications and modifying enzymes
Of the 42 cytoplasmic tRNA species in Saccharomyces cerevisiae, 13 have a
uridine at the wobble position (U34) (Percudani et al., 1997). Of these 13 tRNA
species, tRNA Leu
has an unmodified uridine (Randerath et al., 1979) and
UAG
tRNA ψIleAψ contains a pseudouridine at the wobble position (Szweykowska-
Kulinska et al., 1994). The remaining 11 tRNA species have ncm5U, ncm5Um,
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mcm5U, or mcm5s2U at position 34 (Glasser et al., 1992, Johansson et al., 2008,
Keith et al., 1990, Kobayashi et al., 1974, Kuntzel et al., 1975, Lu et al., 2005a,
Smith et al., 1973, Yamamoto et al., 1985).
Multiple synthesis steps and gene products are required to form U34 modified
nucleosides. Thirteen gene products are required for the first step in the mcm5/ncm5
side chain formation and 11 to form the s2 group in mcm5s2U (Fig.2). In strains
with a deletion of any of the ELP1-ELP6, KTI11, KTI12, KTI14, SIT4 or SAP185
and SAP190 genes, formation of mcm5U, mcm5s2U and ncm5U nucleosides is
abolished, whereas a deletion of KTI13 dramatically reduces levels of these
nucleosides (Huang et al., 2005). No intermediates of mcm5U， and ncm5U have
been detected in any of the mutants, but s2U is present in tRNAs normally
containing mcm5s2U (Huang et al., 2005, Huang et al., 2008).
Figure 2. Gene products required
for
formation
of
5
5
methoxycarbonylmethyl (mcm ), 5carbamoylmethyl (ncm5) and 2-thio
(s2) side groups on uridines at the
wobble
position
(Adapted
from
Karlsborn et al. (2014b) ). AcetylCoA acts as a donor in formation of
the cm5 side group. Trm9p and
Trm112p
utilize
AdoMet
(S-
Adenosylmethionine) as a methyl
donor to form the mcm5 side group.
The last step in formation of ncm5 is
unknown (?). R represents ribose and
uridine side groups cm5, mcm5, ncm5
and s2 are highlighted in red.
In addition, the tRNA methyltransferase complex Trm9p/Trm112p is required
for the last step in mcm5U formation, but no gene product responsible for the last
step in ncm5U formation has been identified (Fig. 2) (Kalhor & Clarke, 2003,
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Mazauric et al., 2010, Chen et al., 2011a, Leihne et al., 2011). The thiolation
reaction has been reconstituted in vitro and the 11 gene products known to be
required for formation of the 2-thio group present in tRNA Lys
,
mcm5s 2 UUU
, and tRNA Glu
are shown in Figure.2 (Nakai et al., 2008,
tRNA Gln
mcm5s 2 UUG
mcm5s 2 UUC
Leidel et al., 2009, Noma et al., 2009). In S. cerevisiae, presence of a mcm5 side
chain at U34 is essential for efficient 2-thio group formation and the S. pombe
elp3/sin3 mutant lacks both mcm5 and s2 groups of mcm5s2U modification (Nakai
et al., 2008, Leidel et al., 2009, Noma et al., 2009, Bauer et al., 2012).
1.2.2 tRNA modifying enzyme required for m1A58 formation
The presence of 1-methyladenosine (m1A) at position 58 in the TΨC loop has
been reported in tRNAs from all three kingdoms (Björk et al., 1987), suggesting an
evolutionarily conserved role of m1A58 in tRNA structure and function. In S.
cerevisiae, m1A58 is present in 21 tRNA species including tRNA iMet (Jühling et al.,
2009). Trm6p forms a complex with Trm61p in the nucleus and this two-subunit
complex is the tRNA m1A methyltransferase of S.cerevisiae (Anderson et al., 1998,
Calvo et al., 1999, Anderson et al., 2000). Deletion of TRM6 or TRM61 is lethal
unless tRNA iMet is overexpressed. (Anderson et al., 1998). It has been proposed that
m1A58 contributes to the tertiary interactions between the TΨC and D loops of
tRNA iMet (Calvo et al., 1999). Trm6p is required for tight binding of the tRNA
substrate and Trm61p has a conserved motif for binding S-adenosyl methionine (SAdoMet). Like most other tRNA methyltransferases (MTases), the Trm6p/Trm61p
complex uses AdoMet as the methyl donor in m1A formation in yeast tRNAs
(Anderson et al., 2000).
1.2.3 tRNA surveillance
The levels of pre- tRNA iMet are indistinguishable between the trm6-504 mutant
and wild type. However, the amount of mature tRNA iMet is two-fold lower in the
trm6-504 strain (Anderson et al., 1998). Further studies have shown that a tRNA
surveillance pathway – consisting of the exosome and the poly (A) polymerase
Trf4p – present in yeast degrades hypomodified pre- tRNA iMet . The exosome is a
multi-protein complex that occurs in nuclear and cytoplasmic forms, both of which
are capable of degrading various types of RNA (Chlebowski et al., 2013). The
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nuclear form is associated with Rrp6p, a nuclease with 3’-5’ exonuclease activity.
Deletion of the RRP6 gene in a trm6-504 mutant suppresses its temperaturesensitive growth phenotype (attributable to the defect of forming m1A58 and
consequential tRNA iMet instability) and increases mature tRNA iMet levels, strongly
supporting the hypothesis that degradation of hypomodified pre- tRNA iMet occurs in
the nucleus (Kadaba et al., 2004). The yeast exosome, of which Rrp44p is a core
component, contains 10 predicted 3’-5’ exohydrolases or phosphorylases involved
in numerous RNA processing events (van Hoof & Parker, 1999, van Hoof &
Parker, 2002). A mutated allele of RRP44 suppresses the growth defect of a trm6504 mutant and restores the level of tRNA iMet close to normal, corroborating the
exosome-dependence of hypomodified pre- tRNA iMet degradation (Kadaba et al.,
2004). A mutated allele of TRF4 has been identified as a suppressor of the trm6504 mutant (Kadaba et al., 2004). Trf4p exhibits RNA poly (A) polymerase
activity in vitro (Saitoh et al., 2002) and catalyses polyadenylation of
hypomodified pre- tRNA iMet prior to its degradation by the nuclear exosome
(Kadaba et al., 2004).
1.3 Translation
1.3.1 The translation process
The translation process is divided into four stages: initiation, elongation,
termination and ribosome recycling (Kapp & Lorsch, 2004). In eukaryotes,
translation initiation is usually dependent on recognition of the mRNA 5’ cap
followed by initiation complex assembly. However, internal ribosome entry site
(IRES) can also be used as the translation initiation site without ribosome scanning
from the 5’ cap of the mRNA (Lopez-Lastra et al., 2005). In an early step of 5’
cap-dependent initiation of translation, eukaryotic initiation factor eIF2， tRNA iMet ,
and GTP form a ternary complex. With facilitation by eIF1, eIF1A and eIF3, the
ternary complex joins the small (40S) ribosomal subunit, generating a 43S preinitiation complex. With the help of eIF4F complex (consisting of eIF4A, eIF4G,
and eIF4E proteins), the 43S pre-initiation complex is loaded to mRNA and begins
to scan the mRNA in the 5’ to 3’ direction for the translation start codon. When it
reaches an AUG codon, normally the first AUG in a favourable sequence context
(e.g. the Kozak sequence), codon-anticodon base pairing occurs between the AUG
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codon and the tRNA iMet . This triggers hydrolysis of the GTP bound to eIF2 (a
reaction catalysed by eIF5) and the release of tRNA iMet into the P site of the 40S
ribosomal subunit. After dissociation of eIF2-GDP and eIFs (such as 1, 1A, 3, and
5) from the pre-initiation complex, the large (60S) ribosome joins the 40S∙ tRNA iMet
∙mRNA complex. The initiation of translation ends with the GTP hydrolysis on
eIF5B and its subsequent dissociation from initiation complex (Gebauer & Hentze,
2004, Kapp & Lorsch, 2004, Jackson et al., 2010).
In the elongation step, a ternary complex containing an aminoacyl tRNA, eEF1A,
and GTP enters the ribosomal A site. Base pairing between the mRNA and the
tRNA, conformational changes in the decoding centre of the small ribosomal
subunit, and GTP hydrolysis by eEF1A ensure that only the cognate tRNA is
selected for entry into the next stage of elongation (Rodnina & Wintermeyer, 2001).
After GTP hydrolysis, eEF1A∙GDP releases the aminoacyl tRNA into the A site. In
a step catalysed by the peptidyl transfer centre, the peptide present in the peptidyltRNA is transferred and forms an ester bond with the incoming amino acid present
in the aminoacyl-tRNA. Elongation factor 2 (eEF2) facilitates the translocation so
that the deacylated tRNA is in the E site, the peptidyl tRNA in the P site, and the
mRNA moves three nucleotides, thereby placing the next codon in the A site. The
elongation cycle is repeated until a stop codon is present in the A site, leading to
translation termination (Kapp & Lorsch, 2004). In eukaryotes, all three stop codons
are recognized by the class I release factor eRF1. The other release factor eRF3
binds to eRF1, generating an eRF1/eRF3/GTP complex that binds to the ribosomal
A site. Release factor 3 hydrolyses GTP and eRF1 hydrolyses the polypeptidyltRNA, releasing the completed protein product (Jackson et al., 2012, von der Haar
& Tuite, 2007). After translation termination, ribosome subunits are recycled, a
process catalysed by eEF3, Rli7 and ATP in yeast (Kurata et al., 2010, Young et al.,
2015).
1.3.2 Translation errors and frameshifting
The translation machinery decodes mRNAs with high efficiency and fidelity,
although errors occur at a low frequency (Kurland, 1992). A missense error only
changes a single amino acid, and thus does not severely impair the resulting
protein’s functions or stability, unless it occurs in a critical position. However,
frameshift errors are generally detrimental, as the ribosome frequently encounters a
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stop codon in the new reading frame, causing premature translational termination.
Frameshift errors are around 10-fold less frequent than missense errors (Parker,
1989, Kurland, 1992).
A well-established peptidyl-tRNA slippage model explains how tRNA
modification deficiency may induce frameshifting errors (Farabaugh, 1996a,
Farabaugh, 1996b, Björk et al., 1999, Farabaugh & Björk, 1999, Gallant et al.,
2000, Urbonavičius et al., 2001, Atkins & Björk, 2009, Näsvall et al., 2009, Jäger
et al., 2013, Björk & Hagervall, 2014). According to this model (Fig. 3) a
modification-deficient aminoacyl-tRNA present in a tRNA∙eEF1A∙GTP ternary
complex, induces frameshifts either by an A- or a P-site effect, or a combination
thereof. Lack of modification causes a defect in the cognate aminoacyl-tRNA
selection step, allowing acceptance of a ternary complex with a near-cognate wildtype aminoacyl-tRNA in the A-site. After translocation to the P-site, the fit of the
near-cognate peptidyl-tRNA is not optimal, resulting in a slip one nucleotide
forward (+1 frameshift). This is denoted an A- site effect (Fig. 3A). Alternatively,
lack of a modified nucleoside reduces the efficiency of entry of a cognate
aminoacyl-tRNA to the A-site, which induces a ribosomal pause, allowing the wild
type peptidyl-tRNA to slip one nucleotide forward. This is also denoted an A-site
effect (Fig. 3B). When frameshifting is caused by a P-site affect, the hypomodified
tRNA is efficiently accepted to the A-site, and translocated to the P-site where its
fit is not optimal, so it slips into an alternative reading frame (Fig. 3C) (Björk et al.,
1999, Farabaugh & Björk, 1999, Urbonavičius et al., 2001, Atkins & Björk, 2009,
Näsvall et al., 2009). Thus, in some cases, the modification deficiency reduces the
rate of aminoacyl-tRNA selection (A-site effect) but can also reduce the ribosomal
grip in the P- site (P-site effect). Note, in all cases explained above, the error in
reading frame maintenance is due to a peptidyl-tRNA slippage.
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Figure 3. Dual-error frameshifting model.
1.3.3 Role of wobble uridine modifications in translation
In vitro, tRNA anticodon stem loops (ASLs) containing either s2 or mnm5 groups
at U34 can bind to the ribosomal A-site and translocate to the P-site when decoding
A- and G-ending codons, but not U- and C-ending codons (Phelps et al., 2004,
Yarian et al., 2000). A crystal structure reported by Murphy et al. (2004) indicates
that the presence of mnm5U34 or s2U34 modified nucleosides in LysUUU tRNA
improves reading of both AAA- and AAG- ending codons, while NMR data
presented by Sundaram et al. (2000) suggest that mcm5s2U34 containing tRNA
recognizes codons in the same manner as mnm5s2U34-containing tRNA. In addition,
an in vivo study has showed that mcm5 and ncm5 side chains facilitate decoding of
G-ending codons and that simultaneous presence of mcm5 and s2 groups improves
reading of both A- and G-ending codons (Johansson et al., 2008).
A strain lacking mcm5/ncm5 side chains at U34 (e.g. in the elp3Δ mutant) shows
Lys
Gln
pleiotropic phenotypes. Overexpression of hypomodified tRNA s2UUU and tRNA s2UUG
suppresses most of these effects, including perturbations of Pol II transcription and
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exocytosis, but not the tRNA modification defects (Esberg et al., 2006), indicating
that the pleiotropic phenotypes of wobble uridine modification-deficient mutants
are caused by translation defects. It has been shown that both mcm5 and s2 groups
at U34 are important for efficient translation of mRNAs enriched in AAA, CAA,
Lys
and GAA codons, and enhance binding of tRNA mcm 5 s 2 UUU to the ribosomal A-site
(Rezgui et al., 2013). It was also recently shown that in the absence of mcm5, s2 (or
both) side groups of U34 translation pauses when AAA, CAA, and GAA codons are
in the ribosomal A site (Nedialkova & Leidel, 2015, Zinshteyn & Gilbert, 2013).
Wobble uridine modifications are also required for translational frame
maintenance. For example, the modified wobble nucleoside 5-methylaminomethyl2-thiouridine (mnm5s2U34) present in bacterial tRNA specific for Gln, Lys, and Glu,
is important for proper reading frame maintenance (Brierley et al., 1997,
Urbonavičius et al., 2001, Licznar et al., 2003, Urbonavičius et al., 2003, Maynard
et al., 2012). In eukaryotes, previous study of these phenomena had been limited to
an analysis of the role of the esterified methyl group of mcm5U34 in reading frame
maintenance (Patil et al., 2012). Partly for this reason, in the study reported in
Paper I my colleagues and I (hereafter we) investigated the role of wobble uridine
modifications in +1 translational frameshifting in yeast.
1.4 Elongator complex
1.4.1 Elongator complex in yeast Saccharomyces cerevisiae
The Elongator complex of Saccharomyces cerevisiae was first co-purified with
the hyper-phosphorylated elongating form of RNA polymerase II (Pol II) and
found to be composed of Elp1p, Elp2p, and Elp3p (Otero et al., 1999). Later, Elp4,
Elp5, and Elp6 proteins were shown to form a sub-complex with the Elp1p-Elp3p
Elongator core complex (Krogan & Greenblatt, 2001, Li et al., 2001, Winkler et al.,
2001). Based on Elongator complex’s in vitro histone H3 and H4 acetylation (HAT)
activity and transcription-related phenotypic perturbations of Elongator mutants,
this complex was suggested to be required for elongation of Pol II transcription
(Winkler et al., 2002, Wittschieben et al., 1999). Other roles for the Elongator
complex were subsequently proposed in polarized exocytosis (Rahl et al., 2005),
DNA repair (Li et al., 2009) and formation of 5-methoxycarbonylmethyl (mcm5) or
5-carbamoylmethyl (ncm5) side chains at the wobble position (U34) (Huang et al.,
2005). However, several studies now support the observation by Esberg et al.
13
(2006) that the physiologically relevant function of Elongator in yeast is in
formation of mcm5 and ncm5 side chains at U34 of tRNA (Bauer et al., 2012, Chen
et al., 2011b, Esberg et al., 2006, Nedialkova & Leidel, 2015). The hypothesis was
corroborated by recent demonstrations that the homolog of yeast Elp3p in the
archaea Methanocaldococcus infernus, MinElp3, generates cm5U in the presence of
SAM (S-adenosylmethionine) and acetyl-CoA in vitro (Selvadurai et al., 2014).
Furthermore, the plant Arabidopsis thaliana, the worm Caenorhabditis elegans, the
mouse Mus musculus and humans with mutations Elongator genes show defects in
formation of wobble uridine modifications, indicating a conserved role of
Elongator complex in tRNA modification (Chen et al., 2009, Karlsborn et al.,
2014a, Lin et al., 2013, Mehlgarten et al., 2010).
1.4.2 Regulation of Elongator complex activity
Elp1p is the largest subunit of the Elongator complex. It is phosphorylated and
its de-phosphorylation requires the phosphatase Sit4p and the Sit4p associated
proteins - Sap185p and Sap190p (Jablonowski et al., 2004). Elp1p is hyperphosphorylated in a sit4 null mutant, whereas in the casein kinase hrr25 null
mutant, it is hypo-phosphorylated (Mehlgarten et al., 2009). Hyper- and hypophosphorylated Elp1p are balanced in wild type cells and any interference with this
equilibrium may putatively inactivate Elongator complex (Mehlgarten et al., 2009).
Consequently, Sit4p and Hrr25p might regulate the phosphorylation status of Elp1p
and play antagonistic roles in Elongator complex function (Mehlgarten et al., 2009).
In a recent study, nine in vivo phosphorylation sites of Elp1p were identified and
Hrr25p directly phosphorylates two of them (Ser-1198 and Ser-1202) (AbdelFattah et al., 2015). These data indicate that Elp1p phosphorylation plays a positive
role in tRNA modification.
In addition to phosphorylation, the Elp1p is subject to proteolysis. Both affinity
purification of Elongator complex and western blot analysis have revealed two
major and one minor form of Elp1p (Krogan & Greenblatt, 2001, Fichtner et al.,
2003). LC-MS data indicate that an N-terminal truncation causing loss of about
200 amino acids is involved in generation of the shortest form, the level of which
reportedly increases in the absence of Urm1p or Kti11p (Fichtner et al., 2003).
Urm1p and Kti11p have suggested links with Elongator function, as strains with
mutations in genes encoding them (and Elongator mutants), are resistant to
zymocin, a Kluyveromyces lactis toxin (Huang et al., 2008, Fichtner & Schaffrath,
14
2002, Frohloff et al., 2001). It is now clear that the γ-toxin, a subunit of zymocin, is
Lys
an endonuclease targeting tRNA Glu
, tRNA Gln
, and tRNA mcm 5s 2 UUU (Lu et
mcm 5 s 2 UUC
mcm 5 s 2 UUG
al., 2005b). The endonuclease efficiently cleaves these tRNAs between U34 and U35
when mcm5s2U is present at U34 (Lu et al., 2005b). Kti11p is required to form the
mcm5 side chain and Urm1p to form the s2 group of the mcm5s2U34 nucleoside
(Huang et al., 2008). Resistance to γ-toxin, accumulation of truncated Elp1p, and
inability to form the mcm5 or s2 group of mcm5s2U34, are observed in the absence
of Kti11p or Urm1p, suggesting that both Kti11p and Urm1p affect Elp1p
proteolysis and are required for proper Elongator function/regulation (Fichtner et
al., 2003). In the study reported in paper II, we analysed Elp1p proteolysis and its
physiological relevance.
1.5 The PKC family
Protein kinases play key roles in the signal transduction cascades that mediate
cellular responses to environmental cues. The protein kinase C (PKC) family, one
group of these kinases, participates in signal propagation and distribution. PKCs
are conserved in eukaryotes: a single isoform is present in S. cerevisiae, five in
Drosophila melanogaster and 12 in mammals (Mellor & Parker, 1998). The PKC
family can be divided into four subgroups based on differences in regulatory
domains: the “conventional” PKCs (cPKCs), “novel” PKCs (nPKCs), “atypical”
PKCs (aPKCs) and putative transmembrane serine/threonine-protein kinases
(PKNs). The cPKCs (PKCα, PKCβ and PKCγ) are homologs of budding yeast
Pkc1p (Watanabe et al., 1994). In mammals, the serine/threonine kinase PKCα
participates in cell proliferation, differentiation, and apoptosis through divergent
signalling pathways and its expression usually correlates with tumour formation
and drug resistance (Gravitt et al., 1994, Budworth et al., 1997, Lee et al., 2012).
PKCα plays an important, but imperfectly understood, role in gliomas, that are
primary human brain tumours with tumorigenicity characterized by grades
assigned by the World Health Organization (Louis et al., 2007). Elevated PKCα
levels have been observed in cell lines derived from the most malignant form of
glioma tumor in humans - glioblastoma (GBM) (Misra-Press et al., 1992, Xiao et
al., 1994). However, Zellner et al. (1998) found similar levels of PKCα in freshly
frozen samples of both low- and high-grade human gliomas, so expression patterns
of PKCα in gliomas and its role in tumorigenic processes are still controversial
(Mandil et al., 2001, Cameron et al., 2008, Ahmad et al., 1994). In the study
15
described in Paper III, an interaction partner of PKCα in the glioma cell line C6
was identified and effects of their interaction on tumorigenesis were investigated.
16
Results and discussion
Paper I: The role of wobble uridine modifications in +1 translational
frameshifting in eukaryotes
In yeast, the modified wobble nucleoside mcm5s2U34 is present in Gln-, Lys-,
and Glu-tRNA. In bacteria, the related modification mnm5s2U34 is present in the
same set of tRNA species and the modification has been proven to be important for
translational frame maintenance (Brierley et al., 1997, Urbonavičius et al., 2001,
Licznar et al., 2003, Urbonavičius et al., 2003, Maynard et al., 2012). However, in
eukaryotes, limited prior analysis of mcm5U34 functions had focused on the
esterified methyl group’s role in maintaining the proper reading frame, where the
esterified methyl group was shown to be important to prevent -1 frameshifting
(Patil et al., 2012). Thus, to extend knowledge of the modification’s roles, we
investigated whether the ncm5U, mcm5U, or mcm5s2U modifications are crucial for
reading frame maintenance in yeast. We first used the Renilla/Firefly luciferase
bicistronic reporter system to address this question. A small frameshifting window,
consisting of slippery codon, test codon, and in-frame stop codon, was inserted
between Renilla and Firefly genes in such a way that Firefly luciferase expression
is dependent a +1 frameshifting event in the window. The level of frameshifting is
revealed by dividing the ratio of F-luc/R-luc activities generated from the
frameshifting construct by the ratio of activities generated from an F-luc/R-luc inframe control. Comparisons of ratios between wild type and modification-deficient
mutants showed that the mcm5 group plays a more vital role than the ncm5 group in
reading frame maintenance. The s2 group of mcm5s2U also plays an important role
in translational frame maintenance, but presence of the esterified methyl group in
the mcm5 side chain only seems to play a minor role in prevention of +1
frameshifting.
According to the dual-error frameshifting model, frameshifting events are due to
A- or P- site effects. To determine whether the frameshifting we observed is caused
by an A- or a P-site effect, we utilized a modified Ty1 frameshifting site in a
HIS4A::lacZ reporter system. A seven nucleotide (CUU-AGG-C) sequence is
required for the +1 frameshift event that results in expression of TyB of the yeast
Ty1 retrotransposon (Belcourt & Farabaugh, 1990). Two tRNA species, tRNA Leu
UAG
Arg
and tRNA CCU
, are involved in this event. We changed the CUU-AGG-C sequence
17
to CUU-AGA/AAA-C and inserted it into the 5’ end of a lacZ gene in such a
manner that lacZ expression required occurrence of a +1 frameshifting event within
these seven nucleotides. We then determined lacZ expression in the presence or
absence of mcm5U34. Approximately ten-fold higher lacZ levels were observed in
an elp3 null mutant than in wild type controls when the AAA codon was used in
Lys
the test. Overexpression of tRNA s 2 UUU in the elp3 null mutant reduced the lacZ
expression to about 3-fold greater than wild type, strongly indicating that the +1
frameshifting event in the “CUU-AAA-C” Lys codon test construct occurs by
Lys
peptidyl tRNA Leu
UAG slippage due to slow entry of the hypomodified tRNA mcm 5 s 2 UUU .
Thus this frameshifting event is probably caused by an A-site effect.
Wobble uridine modification-deficient mutants show pleiotropic phenotypes, all
of which except the wobble uridine modification defect can be suppressed by
overexpression of hypomodified tRNAs, indicating a defect of hypomodified
tRNAs in decoding (Esberg et al., 2006). Our data suggest that the pleiotropic
phenotypic perturbations of wobble uridine modification-deficient mutants might
be at least partly due to frameshift errors.
Paper II: Yeast Elongator protein Elp1p does not undergo proteolytic
processing in exponentially growing cells
As mentioned above, a previous study indicated that Elp1p, the biggest subunit of
the Elongator complex, is subject to proteolytic processing that generates two
major (~160 kDa and ~140 kDa) and one minor (~120 kDa) form (Krogan &
Greenblatt, 2001), which may be required for proper Elongator function and/or
regulation (Fichtner et al., 2003). There are two in-frame start codons at the 5’ end
of the ELP1 ORF, separated by 48 nucleotides. The 2nd ATG was determined to be
the translational start codon for Elp1p. To analyse the in vivo composition of
Elongator complex, tagged Elp1p was expressed and the complex was purified
using the tandem affinity purification (TAP) procedure. In addition to the three
forms already mentioned, a ~26 kDa fragment representing the N-terminus of
Elp1p was found, strongly indicating that an endopeptidase is required for the
cleavage of Elp1p. We screened 95 non-essential peptidase/protease null mutants
to identify strain(s) unable to form the truncated Elp1p. In the vacuolar protease
prb1 null mutant, no truncated Elp1p was observed, indicating that Prb1p is
18
required for Elp1p proteolysis. Amine-reactive Tandem Mass (TM)-tag followed
by LC-MS/MS analysis showed that Elp1p is cleaved between the 203rd (Lys) and
204th (Ala) residues. The elp1 gene encoding the truncated Elp1p starting from Ala
204 was cloned into a high copy vector and expressed in an elp1 null mutant, but
this did not result in complementation of the wobble uridine modification defect.
An intact six-subunit Elongator complex was subsequently purified from a prb1
null mutant, and levels of wobble uridine modifications in total tRNA from the
mutant were found to be similar to those in tRNA from a wild type strain,
indicating that Prb1p is not required for Elongator complex assembly or tRNA U34
modifications. When protein was extracted from a mixture containing equal
amounts of harvested exponentially growing elp1Δ and prb1Δ strains, a truncated
Elp1p was detected, implying that cleavage occurs in vitro during sample
preparation. To avoid any potential endopeptidase-mediated cleavage in vitro and
determine whether the truncated Elp1p is present in a wild type strain, we included
TCA in the protein extraction solution to ensure that extracted proteins were
precipitated immediately after cell lysis. Using this method, no truncated Elp1p
was observed in the elp1Δ/prb1Δ mixed sample. In addition, only full-length Elp1p
was detected in the wild type strain. When TCA was excluded from the protein
extraction solution, more truncated Elp1p was detected in kti11Δ and urm1Δ strains,
but appearance of this truncated fragment was abolished in the presence of TCA.
Taken together, these findings clearly show that the truncated Elp1p is a
preparation artifact.
Paper III. TRM6/61 connects PKCα with translational control through
tRNA Met
stabilization: impact on tumorigenesis
i
PKCα is a serine/threonine kinase that plays a vital role in diverse signalling
pathways involved in cell proliferation, differentiation and apoptosis. However, the
relationships between PKCα expression patterns and aggressiveness of tumours are
controversial. Thus, to assist efforts to clarify its roles, we sought new interaction
partner(s) for PKCα in glioma cells and identified TRM61. TRM61 forms a
complex with TRM6 and the complex has methyltransferase activity modifying A58
in tRNA to m1A58 (Ozanick et al., 2005). In the glioma cell line C6, PKCα
localizes in cytoplasm and TRM6 in the nucleus, whereas TRM61 is present in
both compartments. In yeast, Trm61p has a predicted nuclear localization signal
19
(NLS) and Pkc1p interacts with Trm6p instead of Trm61p. RNA silencing of
TRM6, TRM61, or both resulted in significant reduction of cell growth
accompanied by tRNA iMet destabilization. Increasing the level of tRNA iMet , but not
tRNA eMet , rescued the growth defect of TRM6/61-depleted cells.
To test whether TRM6/61 activity could influence protein synthesis rate or
favour translation of specific mRNAs, the two proteins were overexpressed. This
resulted in increased tRNA iMet and tRNA eMet levels, and the entire tRNA iMet pool has
m1A58 modification. However, high TRM6/61 levels only slightly increased global
translation and the polysome profile remained very similar. Analysis of mRNAs
extracted from heavy polysome fractions detected significant changes in mRNA
occupancy of 257 transcripts following TRM6/61 overexpression (increased
occupancy of 158 and declined occupancy of 99). Comparison of total cellular
mRNA and polysomal fractions of some genes corroborated significant adjustment
of gene expression at the translation level.
Gliomas are characterized by their tumorigenicity: grade II designates low
tumorigenicity, Glioblastoma multiforme (GBM) is the most aggressive state and
grade III is intermediate. We found higher tRNA iMet and tRNA eMet levels in GBM
samples than in pooled grade II/III patients’ samples. TRM6 and TRM61 mRNAs
levels were also elevated in GBM samples, suggesting that increased tRNA
methylation activity contributes to tRNA iMet accumulation during tumour
progression. We also examined effects of either TRM6/61 or tRNA iMet
overexpression on oncogenic transformation at the cellular level, and found that it
significantly affected cells’ anchorage-independent growth on soft agar, as well as
cell self-renewal. Furthermore, the effects were enhanced when both TRM6/61 and
tRNA iMet were overexpressed, indicating that tRNA iMet overexpression and its
stabilization by m1A58 modification have synergetic effects.
In addition, overexpression of PKCα in a TRM6/61/ tRNA iMet overexpression
background downregulated tRNA iMet levels, reduced cell growth rates, and changed
the intracellular distribution of TRM61 (reducing the nuclear pool and increasing
its cytoplasmic pool, with a concomitant accumulation of PKCα in cytoplasm).
TRM61 sequestration likely reduces TRM6/61 activity and prevents abnormal
translation inducing tumor progression. Accordingly, analysis of patient samples
20
revealed declines in PKCα mRNA levels during the course of cancer progression,
correlating with increases TRM6/61 mRNA levels. Taken together, these findings
indicate that tightly control of TRM6/61 activity by PKCα is required to prevent
translation perturbations that can induce neoplastic development.
21
Conclusions
1. Presence of mcm5 and s2 side groups at wobble uridines is important for
reading frame maintenance. The +1 frameshifting observed in the Lys
codon test construct occurs via peptidyl-slippage due to an A-site effect.
2. Elp1p is cleaved between residues 203 (Lys) and 204 (Ala) by vacuolar
protease Prb1p. N-terminal truncation of Elp1p is a preparation artifact,
and thus is highly unlikely to regulate Elongator complex activity.
3. In glioma cell line C6, PKCα interacts with TRM61. PKCα overexpression
reduces nuclear levels of TRM61, likely suppressing TRM6/61 activity.
Comparison of highly aggressive glioblastoma multiforme (GBM) to
Grade II/III glioblastomas indicates that reduction in PKCα expression is
related to increased levels of TRM6/61 transcripts and tRNA iMet ,
highlighting the clinical relevance of the study.
22
Acknowledgements
Above all, I strongly appreciate my initial acceptance by my supervisor Anders
Byström as a PhD student. You gave me the chance to explore biology from
scratch, which is not the easiest thing to do on earth. Your curiosity about the
results has fuelled my motivation, and your rigorous attitude to writing reminds me
of the sanctity of science. I truly admire you as a scientist. Many thanks to Mona
for nice diners you made, and for forcing Anders to have vacations sometimes,
which he doesn’t want but he and his students really need.
My co-supervisor Marcus, you are such a smart human being, with great lab
experience, and I’m so impressed by your critical thinking. I strongly believe that if
you couldn’t be a successful scientist, then no one could. Plenty of gratitude is also
due to the current and previous members of the ABY group: Hasan, you probably
have the most organized lab bench in this department (and maybe the world?) and
without you the frameshifting paper would never have reached its current state.
Tony, you represent all the best of robust Swedish education, displaying broad
knowledge and great skills in labs; you would be perfect if you could be more
cautious when handling phenol. Fu, you embody the diligent and determined spirit
of Fudan University, best wishes for your projects as well as the real estate
business in Umeå. Gunilla, thanks for all the professional help you provide and all
the fancy dinners at your place. Look at your beautiful pictures in Facebook, you
are an artist. Changchun, my tutor, thank you for teaching me almost everything in
the lab, from setting parameters for a centrifuge to the tricks for making a flawless
tRNA northern gel. Firoj, thanks for the suggestion about statistics and
congratulations again on becoming a father. Anders E., thank you for initiating the
frameshifting project.
I would like to thank Gunnar and Joakim for your collaborative efforts with us.
Special thanks to Glenn, you are the idol of our field and I appreciate your reading
of our manuscripts and valuable suggestions. I also thank Tracy and your group
members for valuable discussion in the journal club meetings. Yang, thanks a lot
for lending me things I had ordered but hadn’t come yet. Lina, Ala, and
Ümmühan, it’s a pleasure to teach courses with you. Marie-Line, you are always
welcome in our office for some morale-boosting chat if you get bored. Many
thanks to all the nice people in the Department of Molecular Biology: Sa, Erik,
Margarida, Aman, Junfa, Edvin, Stefanie, Shahab, Lisa, Helen, Congting and
23
so on and so on… I wish you all the best! I am also very grateful to Johnny,
dishwashing, media and secretaries for offering priceless services, making our
life much easier.
I am so thankful to Umeå for bringing me many friends! Xingru, I appreciate all
the happy time we spent together! Thank you so much for introducing Işil, Lee
Ann, Robert and Greg to me. All of you are great people and I have a lot of fun
every time when we hang out (or sometimes hangover?). The trip to Hong Kong
was as cute as those pandas we saw in the Ocean Park. Linlin, hiking to Pulpit
Rock in Norway with you will be high point of my life for a long time as you can
see from my profile picture (which I haven’t changed since then). Xi, you know I
am a big fan of you and can’t wait to get back to Shanghai to bother you and be
bothered by you. Hairu, I appreciate your generosity in never getting mad at me
for making fun of you and your sincerity is a pure treasure. Binbin and Kunkun, it
is a shame I came to know you so late, but there is an entire future ahead of us,
right? Dan, I appreciate your decency, patience and company. You are welcome to
visit Shanghai at any time. Yutong, Sunkun, Zihao, Xintao, Siyuan and all the
people I met in Umeå, thank you all for the good time we had!
Dear Prof. Jin, it is said that a few people are destined to change one’s life. I am
sure that you are that kind of person for me. Thanks for believing in me and giving
me a chance to become a better version of myself.
感谢爸爸，你一直都是我世界里最稳定的依靠，我年纪越大越是能体会到你的闪光，很
爱你。谢谢柳姿还有柳姿的父母，我和柳姿会尽量努力让你们放心的。最后，写几句给妈妈
的话：当初出国念书的决定多少伤害了你，如今毕业的时刻你也无法亲眼目睹，人生用最严
厉的方式真真实实地书写了遗憾二字。不过答应你的我已经做到，我也一直明白生者乐观豁
达的幸福生活才是对逝者最好的慰藉和纪念，我会尽力，这是对你新的承诺。
24
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