RNA DECAY
PART I - GENERAL MECHANISMS
PART II - SPECIFIC PATHWAYS
REGULATION OF GENE EXPRESSION
1) PolII assembly
2) transcription
3) mRNA processing
4) mRNA export
5) translation
6) protein stability
7) RNA degradation
RNases
Endonucleases
processing (RNase P, RNase III, RNase E):
specific, cleavage results in 3’-OH and 5’-P (monophosphate)
degrading (RNase I, RNAse A):
unspecific, cleavage results in 5’-OH and 3’-P (cyclic phosphate)
Exonucleases
hydrolytic: attacking group H2O, results in 3’-OH and 5’-P
phosphorolytic: attacking group inorganic phosphate, results in 3’-OH and 5’-PP
distributive
processive
Symmons et al, TiBS, 2002
Prokaryotic RNases
Family
RNases
Exonucleases 3’
Characteristics
5’
RNR
RNase II
RNase R
nonspecific processive, degrades only ssRNA, mRNA decay
nonspecific processive, degrades ssRNA and dsRNA, mRNA decay
DEDD
RNase D
RNase T
Oligoribonuclease
distributive, small RNA and stabile RNA processing
specific for oligoribonucleotides
RBN
RNase BN/Z
distributive exonuclease 3’- 5’ and endonuclease, tRNA processing
PDX
PNPase
RNase PH
Exonucleases 5’
*RNAse J1/J2
phosphorolytic processive, degradosome subunit, KH/S1 RNA BD domains, degrades ss/dsRNA
phosphorolytic distributive
3’
present in Bacillus subtilis, specific for 5’ monoP ssRNA, mRNA decay
Endonucleases
RNase III
RNase E
dsRNA specific, rRNA, tRNA, mRNA processing, mRNA degradation
RNase G
RNase I
RNase H
RNase P
RNase Z
similar to RNase E
MazF/EndoA
RNAse M5
*RNAse J1/J2
degradosome subunit, mRNA decay; rRNA tRNA and RNaseP RNA processing
nonspecific, mRNA degradation
specific for RNA:DNA hybrid
tRNA 5’ end processing
tRNA 3’ end processing
toxin, mRNA degradation in stress conditions, sequence specific
Mackie, Nat. Rev. Microbiol, 2012
Degradation of bacterial mRNAs
Degradosome - major complex involved in mRNA decay in bacteria, functions as dimer
RNase E
PNPase
RhIB
Enolase
additional
5’-phosphate -dependent endoribonuclease, N-terminal nucleolytic domain, C-terminal
protein binding domain, central RNA binding domain (BD)
phosphorolytic processive exonuclease 3’ - 5’, KH and S1 RNA BD
ATP-dependent helicase, DEAD box, stimulate degradation of structured RNA regions
glycolytic enzyme
DnaK/GroEL chaperons , poliphosphate kinase, poly(A) polymerase, S1 ribosomal protein
Catalytic domain
RNase E
RNA BD
(RRD) C-terminal
NH2
COOH
PDX domain
PNPaza
RhIB
RNA BD RNA BD
(KH)
(S1)
PNPaza
PNPazy trimer
Symmons et al, Structure, 2000
Symmons et al, TiBS, 2002
Degradation of bacterial mRNAs
3’ end stem-loop structure of transcripts targeted for degradation becomes
often
polyadenylated
by
PAP
(poly(A)
polymerase)
and
PNPase
(polynucleotide phosphatase), with the help of Hfq (hexameric RNA chaperone).
RNase E cleavage initiates degradation by 3’ - 5’ exonucleases, mainly
RNase II, RNase R and PNPase.
PNPaza
Hfq
PAP I
Mohanty et al, Mol. Microbiol., 2004
Symmons et al, TiBS, 2002
Protein
Exonucleases 5’
Xrn1
Rat1
Rrp17/Nol12
3’
Function
Eukaryotic RNases
cytoplasmic, mRNA degradation
nuclear, pre-rRNA, sn/snoRNA, pre-mRNA processing and degradation
nuclear, pre-rRNA processing
Exosome 3’
5’ multisubunit exo/endo complex
Rrp44/Dis3
catalytic subunit
Rrp4, Rrp40
pre-rRNA, sn/snoRNA processing, mRNA degradation
Rrp41-43, 45-46
participates in NMD, ARE-dependent, non-stop decay
Mtr3, Ski4
Rrp6; Rrp47p
nuclear helicase cofactor
Ski2,3,7,8
cytoplasmic exosome cofactors
Other 3’ 5’ exonucleases
Rex1-4
3’-5’ exonucleases, rRNA, snoRNA, tRNA processing
mtEXO 3’
Suv3/ Dss1
Characteristics
5’
mitochondrial degradosome RNA degradation in yeast
helicase/ 3’-5’ exonuclease
Deadenylation
Ccr4/NOT
Pop2
Pan2p/Pan3
PARN
major deadenylase complex (Ccr, Caf, Pop, Not proteins)
deadenylation regulator, deadenylase activity
additional deadenylases (poliA tail length)
mammalian deadenylase
subunits organized as in bacterial PNPazy
Exo/PIN domains, distributive, hydrolytic
RNase D homolog; RNA binding
DEAD box helicase, GTPase
RNase D homolog
DExH box/ RNase II homolog
Ccr4- Mg2+ dependent endonuclease
RNase D homolog
RNase D homolog, poly(A) specific nuclease
RNase D homolog, poly(A) specific nuclease
Endonucleases
RNase III
-Rnt1
-Dicer, Drosha
Ago2 Slicer
Utp24
Nob1
SMG6
RNase P
RNase MRP
RNase L
ELAC2/Trz1
pre-rRNA, sn/snoRNA processing, mRNA degradation
siRNA/miRNA biogenesis, functions in RNAi
mRNA cleavage in RNAi
early 35S pre-rRNA processing
18S pre-rRNA processing
mRNA cleavage in NMD
5’ tRNA end processing
pre-rRNA processing
rRNA degradation in apoptosis
3’ tRNA endonuclease
dsRNA specific
PAZ, RNA BD, RNase III domains
PIN domain
PIN domain
PIN domain
RNP complex
RNP complex, similar to RNase P
oligo 2-5A dependent (ppp(A2’p)nA)
PDE motif and Zn2+ -binding motif
Eukaryotic auxiliary decay factors
Function / Characteristics
Protein
5’
3’ decay: decapping
Dcp1/Dcp2
Hedls/Ge-1/Edc4
Edc1,2,3
Dhh1
Lsm1-7
Pat1
Dcp2- pyrophosphatase catalytic activity, Nudix domain, Dcp1- protein binding
decapping cofactor, WD40 domain
decapping enhancers, stimulate cap binding/catalysis, Edc1-2 (yeast), Edc3 (all eykaryotes)
DexD/H ATPase, decapping activator by translation repression
decapping activator, heptameric complex, binds mRNA 3’ end-U rich tracts
decapping activator by translation repression
TRAMP complex: nuclear RNA surveillance, polyadenylation-dependent degradation
Trf4/Trf5
Mtr4
Air1/Air2
nuclear alternative poly(A) polymerases
DEAD box helicase
RNA binding proteins, also nuclear exosome cofactor
Nrd1-Nab3-Sen1 complex: PolII termination of small RNAs, TRAMP-depdendent degradation
Nrd1
Pol II C-terminal domain (CTD) binding, RNA binding
Nab3
Sen1
RNA binding
RNA helicase
RNases
Stoecklin and M. hlemann, BBA, 2013
EXOSOME: 3’ 5’ decay machinery
Crystal structure of human core exosome
Liu et al, Cell, 2006
Dis3
Dziembowski et al, Mol.Cell, 2008; Nature, 2008
• 3’ 5’ exo/endo nuclease complex;
• 10 core components (RNA BP)
• catalytically active exo hydrolytic Dis3/Rrp44 (RNase II)
• PIN domain with endo activity
• nuclear cofactors- RNA BP Rrp47, nuclease Rrp6 (RNase D), RNA helicase Mtr4
• cytoplasmic cofactors- Ski2-3-8 complex (RNA helicase Ski2), GTPase Ski7
• subtrates- processing and/or degradation of almost all RNAs
EXOSOME: 3’
5’ decay machinery: FUNCTIONS
NUCLEAR: Rrp6 and core components have partly separate functions
• 3’ end processing of 5.8S rRNA, sn/snoRNAs, tRNAs, SRP RNA
• degradation of pre-mRNAs, tRNAs, sn/snoRNAs
• degradation of other ncRNAs: CUTs, PROMPTS
CYTOPLASMIC:
• generic mRNA decay
• specialised mRNA decay pathways: NMD, NSD, NO-GO decay, AREdependent decay
XRN family: 5’
3’ processive exonucleases
Kastenmayer and Green, 2000, PNAS
Crystal structure of S. pombe
Rat1/Rai1 complex
NUCLEAR
Rat1/XRN2 with Rai1 activator (5’-ppp pyrophosphohydrolase
and phoshodiesterase-decapping nuclease)
• 5’ end processing of 5.8S and 25S rRNAs, snoRNAs
• degradation of pre-mRNAs, tRNAs, sn/snoRNAs
• degradation of some ncRNAs: CUTs
• transcription termination of Pol I and II (torpedo mechanism)
CYTOPLASMIC XRN1
• generic mRNA decay
Xiang et al, 2009, Nature
• specialised mRNA decay pathways: NMD, NSD, NO-GO decay,
ARE-dependent decay
• degradation of miRNA-dependent mRNA cleavage products (in plants)
• degradation of some ncRNAs: CUTs, SUTs, XUTs
DCP- decapping enzymes
Dcp1p
• Dcp1/Dcp2 complex participates in mRNA 5’ decay
• catalyses the reaction m7GpppX-mRNA
m7GDP + 5’p-mRNA
• Dcp2 is the catalytic subunit (pyrophosphatase Nudix domain)
• Dcp1 is required for activity in vivo, interacts with other proteins
• Dcp1/Dcp2p is regulated by Pab1 and activating factors
(yeast Lsm1-7, Dhh1, Pat1, Edc1-3, Upf1-3)
She et al. Nat.Struct. Mol. Biol, 2004
Dcp2p
Wang et al. PNAS, 2002
DcpS
DcpS
• DcpS: HIT pyrophosphatase („histidine triad” on the C-terminus)
• catalyses the cleavage of m7GDP
m7GMP + Pi remaining after
decapping during mRNA 5’ decay
• cooperates with the exosome during mRNA 3’ decay
(m7GpppX-oligoRNA
Gu et al., M.Cell, 2004
m7GMP+ pp-oligoRNA)
• functions as an asymmetric dimer
LSM PROTEINS
Sm motif
Achsel et al, EMBO J, 2001
Involved in pre-mRNA splicing
• associates with U6 snRNA
• required for U6 RNA accumulation and
U6 snRNP biogenesis
nuclear
• interacts with the U4/U6.U5 tri-snRNP
Functions in mRNA decapping and decay
• activator of decapping
• interacts with components of the mRNA decapping
and degradation machinery (XRN, DCP, Pat1)
cytoplasmic
EUKARYOTIC mRNA
NUCLEUS
CBP80
5’UTR
PABP2
INTRON
AUG
m7GpppG
SPLICEOSOME
5’ ss
CBP20
3’UTR
UAA
3’ ss
AAAAAAAAAAAAA
50-200 nts
CYTOPLASM
EJC
eIF3
PABP1
5’UTR
m7GpppG
eIF4E
3’UTR
AUG
eIF4G
m7Gppp
ORF
UAA
AAAAAAAAAAAAA
50-200 nts
AUG
eIF4E
eIF4G
A
Pab1p
A
A
A
A
A
A
UAA
mRNA t1/2 = few minutes to 2 hours (yeast)
to >90 hours (mammals)
UTR- UnTranslated Region
mRNA DEGRADATION in the CYTOPLASM
DEADENYLATION
RELEASE OF RIBOSOMES
the
RELEASE OF TRANSLATION
FACTORS
he
RECRUITMENT OF DECAY
FACTORS (decapping)
t
P-BODY ASSEMBLY
RNA DEGRADATION
or storage?
Coller and Parker, Annu. Rev. Biochem., 2004
NORMAL mRNA DECAY
IN THE CYTOPLASM
Pab1p
eIF4G
eIF4E
m7Gppp
AAAAA…..AAAAA
Pat1p
NORMAL mRNA DECAY
IN THE CYTOPLASM
dissociation
m7Gppp
Pat1p
AAAAA…..AAAAA
Ccr4/Pop2/Not
Pan2/Pan3
deadenylacja
deadenylation
NORMAL mRNA DECAY
IN THE CYTOPLASM
Recruitment of
Lsm1-7 Lsm/Dcp
m7Gppp
AAAA…AAA
Pat1p
NORMAL mRNA DECAY
IN THE CYTOPLASM
5’-3’degradation
decapping
Lsm
EgzosomC
Xrn1p
AAAA…AAA
Pat1p
Ski2p
3’-5’degradation
• normal mRNA decay involves deadenylation
• LSM/Pat1 binds and protects deadenylated mRNA 3’ ends against
3’-5’ degradation and recruite Dcp complex to activate 5’-3’ decay
• depending on organism different pathway (5’-3’ or 3’-5’) dominates
mRNA DEGRADATION in the CYTOPLASM
mRNA DEGRADATION
in P-BODIES
mRNA DEGRADATION
on POLYSOMES
Balagopal and Parker, Cur.Op.Cel.Biol., 2009
P bodies- processing bodies
(decay bodies, DCP bodies, GW bodies)
P- bodies:
• cytoplasmic dynamic structures of mRNA and decay factors storage
(LSM, DCP, XRN, GW182)
• sites of mRNA degradation
hLSM1-GFP
-hDCP1
overlay
SPECIES:
Yeast
Human cells
Drosophila
C. elegans
CONTENT:
Dcp1/2
Lsm
Edc1/2/3
Dhh1, Pat1
SMG5-7
GW182
Xrn1 siRNA
general
human
C. elegans
AIN-1, ALG-1 miRISC
Cougot et al, JCB, 2004
• mRNA decay factors co-localize and polyA+ RNA accumulates in P bodies
• degradation bodies differ from stress granules
Assembly of P bodies
Mutations in mRNA
decay affect size and
number of P-bodies Aging, glucose deprivation and
osmotic stress increase P-bodies
Dcp2-GFP
Inhibition of translation
activates P-bodies
Dcp2-GFP
prt1ts
prt1ts
-GLU
23C
37C
mRNAs move between
polysomes and P-bodies
Dcp2-GFP
15 min 1M KCl
YEAST
Sheth and Parker, 2003, Science;
Brengues et al, 2005, Science
Franks and Lykke-Andersen, 2008, Mol.Cell
LINK: mRNA DECAY and TRANSCRIPTION
mRNA DECAY and TRANSCRIPTION
mRNA homeostasis
Rpb4/7
Rpb4/7
Rpb4/7
Pat1
Lsm1-7
Perez-Ortin et al, 2013, JMB
RNA DECAY
PART II - SPECIFIC PATHWAYS
RNA SURVEILLANCE =
RNA QUALITY CONTROL MECHANISMS
•
NMD- (nonsense mediated decay) - degradation of mRNAs with
premature stop codons (PTC)
•
NSD- (non-stop decay) - degradation of mRNAs with no stop codons
•
NO-GO decay- degradation of mRNAs stalled in translation elongation
•
AMD - ARE mediated decay- rapid degradation of mRNAs with
specific instability elements (e.g. AU-rich)
•
NRD- Non-functional rRNA decay
•
nuclear RNA degradation (mRNA, pre-mRNA, rRNA, tRNA, ncRNAs)
- degradation of RNA species that were not properly processed i.e.
spliced, end-matured, modified or of unstable species (CUTs)
THE IMPORTANCE OF RNA QUALITY CONTROL
•
these mechanisms control the synthesis, integrity and lifespan of all
cellular RNA molecules to assure optimal functioning of the cell
•
deficient QC and mutations in QC components lead to severe
defects and diseases
- several genetic disorders (30%!- e.g. β-thalasemia, ostegenesis
imperfecta, Marfan syndrome, Stickler’s syndrome, neurologic
syndromes) result from inefficient NMD and other QC mechanisms
due to frameshift mutations and premature translation termination
- mutations in RNA enzymes (exosome, decapping, deadenylases)
confer autoimmune diseases in humans and developmental defects in
plants, worms and flies
NMD
- degradation of mRNAs containing premature STOP codons (PTC)
- prevents expression of truncated, possibly harmful, proteins
- 33% of yeast intron-containing mRNAs undergo NMD
- 30% of alternatively spliced human mRNAs generate NMD substrates
40S
Pab1
Translation termination problem:
- premature termination or
- ribosome stalling on PTC
mRNA degradation
Amrani et al. Nat.Rev.Cel.Biol., 2006
NMD FACTORS
Stalder and Muhlemann, TiCB, 2008
Llorka. Cur. Op. Chem. Biol. 2013
SMG7
Behm-Ansmant et al., FEBS Lett, 2007
EXON JUNCTION COMPLEX (EJC)
metazoan
core
• eIF-4AIII - DEAD box RNA helicase
• MLN51 - stimulates eIF-4AIII
• Magoh/Y14 - heterodimer
binds to eIF-4AIII
Associated factors
• UAP56, SRm160, Acinus,
SAP18, Pinin, RNPS1 - splicing
• REF, TAP, p15 export
• Upf2, Upf3 NMD
EJC contains proteins linked to different functions
MECHANISM OF NMD
1. Recognition of premature stop codon during translation
EJC
PTC
splicing-related mechanism
- EJC deposited as a mark of splicing
- Upf3 is bound to mRNA via EJC
- mRNA is exported and Upf2 joins Upf3
translation termination and unified
3’UTR mechanism:
ribosome not interacting with 3’UTR factors
is arrested on the PTC
2. Assembly of the active NMD complex and repression of translation
EJC downstream of PTC is not removed
by the advancing ribosome
active NMD complex
Upf1-3 + SMG proteins
SURF complex, Upf1.SMG1 and eRF1-2,
is recruited by the stalled ribosome
Upf1 is phosphorylated by SMG1
eRF1-eRF2 are released
mRNA is directed for degradation
Singh and Lykke-Andersen, TiBS 2003; Isken and Maquat, Gene Dev. 2007
MECHANISM OF NMD
3. mRNA degradation
NMD 5’
DCP1/2
3’
deadenylation-independent decapping
NMD endonucleolytic cleavage
XRN1
(Drosophila melanogaster, humans)
exonucleolytic 5’
3’ degradation (XRN1)
decapping is triggered by SMG7 recruitment
or dephosphorylation of Upf1
NMD 3’
5’
egzosomC
recruitment of the Exosome
by Upf1
exonucleolytic 3’
degradation
5’
Parker and Song, Nat. Struct. Mol. Biol. 2004; Isken and Maquat, Gene Dev. 2007
SMG6
NGD and NSD
NGD (non-go decay) - degradation of mRNAs stalled on ribosomes
• NSD- (non-stop decay) - degradation of mRNAs with no stop codons
•
NGD
NSD
strong RNA
structure
stalled
translation
Dom34/Hbs1
ribosome stalls
on poly(A) tail
endonucleolytic
cleavage (?)
Ski7 recruits
the exosome
ExosomeC
Xrn1
Dom34 – has RNA-binding Sm fold
Hbs1- GTPase binding activity
Garneau et al, Nat. Rev. Mol. Cel. Biol. 2007
alternative
pathway
ExosomeC
Xrn1
NGD and NSD
NGD (non-go decay) - degradation of mRNAs stalled on ribosomes
• NSD- (non-stop decay) - degradation of mRNAs with no stop codons
•
Tsuboi et al, Mol. Cell 2012
Dom34:Hbs1 stimulates degradation of the 5’-NGD intermediate and
nonstop mRNA by dissociating the ribosome that is stalled at the 3’ end of
the mRNA
•
AMD ARE - mediated decay
AMD degradation of mRNAs containing AU-rich instability elements
present in mRNA 3’ UTR
ARE-binding proteins
(AUBP)
mRNA instability elements:
- U rich
- AU rich
AUUUA, UUAUUUA(U/A)n
- oligo(U) (or CUCU) tails
- DST (plants)
GGAnnAUAGAUUnnnCAUUnnGUAU
• Exosome (RRP45, RRP41, RRP43) is recruited by ARE-binding proteins AUBP (AUF1)
Roretz and Galouzi, J. Cell. Biol. 2008
TTP
BRF1
HuR
AUF1
KSRP
• Exosomal subunits interact directly with ARE sequences
• PARN and CCR4, deadenylases and XRN1, DCP1/2 interact with AUBP (TTP, KSRP, BRF1)
AMD - connections with RNAi?
The RNAi connection?
Georg Stoecklin’s website
• TTP binds Ago2
• Ago1/Ago2 and Dicer are required for the rapid decay of some ARE-mRNAs
• Cooperation with the RNA-induced silencing complex (RISC) may
contribute to controlling the translation and decay rate of ARE-mRNAs
OTHER MECHANISMS
Endonuclease mediated decay
RNase MPR, RNase III (Rnt1)
IRE1, PMR1
ExosomeC
Xrn1
• PMR1 - degradation of translationally active mRNAs on polysomes
• IRE1 - degradation of mRNAs in endoplasmic reticulum during Unfoded
Protein Response stress
• MRP (RNP responsible for pre-rRNA processing in the nucleolus) - cleaves CLB2
mRNA within its 5’ UTR in yeast
• Rnt1 (RNAse III endonuclease, involved in pre-rRNA and pre-sn/snoRNA processing) cleaves stem-loops structures in some ribosomal protein mRNAs
Garneau et al, Nat. Rev. Mol. Cel. Biol. 2007
OTHER MECHANISMS
Deadenylation-independent decay
(RPS28B and EDC1 mRNAs)
Dcp2
RPS28B
Edc3
Rps28B
Dcp1
Xrn1
Autoregulatory mechanism: Rps28B protein
• binds to a stem-loop structure within the 3’ UTR of its own mRNA
• recruits enhancer of decapping Edc3
• stimulates decapping by Dcp1/2
Garneau et al, Nat. Rev. Mol. Cel. Biol. 2007
mRNA DECAY in the NUCLEUS
• nuclear retention of intron-containing pre-mRNAs
Processing
Quality Control
Export
RES complex (REtention and Splicing): Snu17/Bud13/Pml1
Casolari and Silver, TiCB., 2004
mRNA DECAY in the NUCLEUS
• pre-mRNA with unspliced introns
AAAA…AAA
mm7Rat1p
7 Gppp
7Gppp
m
Gppp
Lsm2-8
ExosomeN
AAAAAAA…AAAAAA
Mtr4p
mRNA DECAY in the NUCLEUS
• mRNA arrested in the nucleus
NUCLEUS
Dcp1/2p
Rat1p
m7G
Lsm2-8
m7G
CYTOPLASM
AAAA…AAA
AAAAAA
Rrp6
mRNA export
blocked
AAAAAA
TRAMP/NEXT EXOSOME COFACTORS
TRAMP =
Trf4/5 +
poly(A)
polyadenylation
polymerases
complex
Air1/2 +
Mtr4
RNA binding RNA DEVH
proteins
helicase
YEAST
Interacts with
- exosome via Mtr4
- Nrd1/Nab3 complex
some RNAs
degraded by
Rat1/Xrn1
HUMAN
Polyadenylation-mediated nuclear
discard pathway for defective RNAs
• hypomodified tRNAs, pre-tRNAs
• ncRNAs:
sn/snoRNAs, rRNAs, some mRNAs
CUTs (Cryptic Unstable Transcripts)
ZCCHC8
Zn-knucle
RMB7
RNA binding
LaCava et al., Cell, 2005; Vanacova et al., PLoS Biol. 2005; Wyers et al., Cell, 2005; Lubas et al. Mol. Cell, 2011
TRAMP + EXOSOME
NUCLEAR RNA SURVEILLANCE
TRAMP
• interacts with the Exosome via Mtr4 - role in degradation
• interacts with Nrd1/Nab3 complex - role in ncRNA Pol II termination
• role in transcription silencing in S. cerevisiae and S. pombe (Cid14)
Vanacova and Stefl, Embo Rep, 2007
TRAMP/EXOSOME - CENTRAL TO ALL
Houseley et al., Nat. Rev. Mol. Cel. Biol. , 2006
Tuttuci and Stutz., Nat. Rev. Mol. Cel. Biol. , 2011
Rat1 - NUCLEAR RNA SURVEILLANCE 5’-3’
Rat1/Xrn2
• decay of transcripts with aberrant
cap structure
• degradation of prematurely
terminated nascent transcripts
• degradation of readthrough
transcripts
Tutucci and Stutz., Nat. Rev. Mol. Cel. Biol. , 2011
CAP NUCLEAR RNA SURVEILLANCE 5’-3’
S. cerevisiae Rai1 – Rat1 activator
5’-ppp pyrophosphohydrolase and phoshodiesterase-decapping nuclease
(unmethylated cap-specific)
CAP NUCLEAR RNA SURVEILLANCE 5’-3’
S. cerevisiae Dxo1:
-phoshodiesterase -decapping
nuclease
- 5’-3’ exonuclease
- no pyrophosphohydrolase
Human DXO
- 5’ppp pyrophosphohydrolase
- phoshodiesterase -decapping
nuclease
- 5’-3’ exonuclease
tRNA SURVEILLANCE
RAPID tRNA DECAY
• occurs for precursors and mature tRNAs with mutations which destabilize
tertiary structure (modifications)
- in the nucleus (polyadenylation via TRAMP and degradation by the exosome or
degradation by Rat1)
- in the cytoplasm (degradation by Xrn1)
Phizicky and Hopper , GeneGev.,2010
tRNA SURVEILLANCE
mature tRNA
(hypo-modified)
pre-tRNA
(hypo-modified)
Interplay between tRNA synthesis and degradation
• tRNA primary transcript synthesized
by RNA Pol III, regulated by Maf1
• Initial processing in the nucleus: 5’
leader and 3’ trailer removed
• pre-tRNA exported to the cytoplasm
• CCA on the 3’ terminus and some
modifications added to pre-tRNA
• Intron spliced out on the outer surface
of the mitochondrial membrane
• tRNA charged by tRNA synthetase,
bound by elongation factor (eEF1A) and
delivered to ribosomes for translation
• tRNA turnover:
(i) in the nucleus pre-tRNAs degraded by
the exosome or by Rat1 in Rapid tRNA
Decay (RTD)
(ii) in the cytoplasm mature tRNAs
degraded by Xrn1-mediated RTD.
• Mature tRNA cleaved into tRNA halves
under stress
Wichtowska, Turowski, Boguta, WIREsRNA, 2013
pre-tRNAs are DEGRADED by the EXOSOME
Dis3
endo
Dis3
exo
Important contribution of the endo Dis3 activity to the degradation
of structured substrates
rRNA SURVEILLANCE
Nucleus:
pre-rRNAs
Nrd1/Nab3
(via pre-mature
termination)
Lafontaine, TiBS.,2010
rRNA SURVEILLANCE
NRD- nonfunctional rRNA decay
Cytoplasm:
mature ribosomes
mutations in peptidyl
transferase centre
Mms1, Rtt101subunits of E3 ubiquitin ligase complex
mutations in
decoding site
Dom34::Hbs1
factors involved in NGD and NSD
Lafontaine, TiBS.,2010
RNA SURVEILLANCE
pre-tRNAs
cytoplasm
nucleus
nucleus
NRD Nonfunctional
rRNA Decay
nucleus
Tuttuci and Stutz., Nat. Rev. Mol. Cel. Biol. , 2011
cytoplasm
nucleus
RNA
SURVEILLANCE
Stoecklin and Mühlemann, BBA, 2013
OLIGO-URIDYLATION
PUP Poly(U) Polymerases
TUTase Terminal Uridylyl Transferase
3’ oligouridylation
1. Histone mRNA degradation (metazoans)
3’hExo
Eri-1
Lsm1-7
3’hExo
Eri-1
3’hExo
Eri-1
Mullen and Marzluff, Genes Dev., 2008
OLIGO-URIDYLATION
PUP Poly(U) Polymerases
TUTase Terminal Uridylyl Transferase
3’ oligouridylation
1. Histone mRNA degradation (metazoans)
3’hExoE
ri-1
3’hExoE
ri-1
3’hExoE
ri-1
Mullen and Marzluff, Genes Dev., 2008
OLIGO-URIDYLATION
2. miRNA degradation
DIS3L2
precursors
C. elegans
mammals
mature
3. mRNA degradation? (plants)
Arabidopsis
Chlamydomonas
Lsm1-7
Krol et al., Nat Rev Genet, 2010; Kim et al., Cell, 2010
STRESS-INDUCED ENZYMATIC
tRNA and rRNA DEGRADATION
(S. cerevisiae, D. melanogaster, A. thaliana, A. nidulans, human cell lines)
Thompson and Parker, Cell, 2009
RNA DECAY
TAKE-HOME MESSAGE
• NORMAL (usually in the cytoplasm)
• SPECIALIZED RNA SURVEILLANCE
targeting aberrant or unstable transcripts for the discard pathway
(NMD, NSD, NGD, ARE, NRD)
1. deadenylation  decapping  exonucleolytic degradation
5’-3’ by Xrn1/Rat1 or 3’-5’ by exosome
2. by endo- cleavage (miRNA-dependent, RNAse III/Rnt1, MRP, SMG6,
Dom34) followed by exo- digestion (Xrn1/Rat1, exosome)
• NUCLEAR RNA SURVEILLANCE
- polyadenylation-mediated by TRAMP (Trf4/5) followed by
degradation by the exosome or Rat1
- related to pre-mature termination or aberrant cap structure by Rat1
• MEDIATED BY OLIGOURIDYLATION
Processing and degradation are often carried out by the same machineries
TAKE-HOME MESSAGE
RNA DECAY
• normal (usually in the cytoplasm)
• specialized, RNA surveillance: targeting aberrant, unstable
transcripts for the discard pathway (NMD, NSD, NGD, ARE, NRD)
1. deadenylation  decapping  exonucleolytic degradation
5’-3’ by Xrn1/Rat1 or 3’-5’ by exosome
2. by endo- cleavage (miRNA-dependent, RNAse III/Rnt1, MRP, SMG6,
Dom34) followed by exo- digestion (Xrn1/Rat1, exosome)
• nuclear RNA surveillance: polyadenylation by TRAMP (Trf4/5)
followed by degradation by the exosome, Xrn1 or Rat1
• post-transcriptional gene silencing
mRNA cleavage
translation inhibition/mRNA decay
Processing and degradation are often carried out by the same machineries