RNA DECAY

RNA DECAY

PART I - GENERAL MECHANISMS

PART II - SPECIFIC PATHWAYS

1) PolII assembly

5) translation

6) protein stability 3) mRNA processing

REGULATION OF GENE EXPRESSION

2) transcription 4) mRNA export

7) RNA degradation

Exonucleases

Symmons et al, TiBS, 2002

distributive

processive hydrolytic: attacking group H

₂

O, results in 3’-OH and 5’-P

phosphorolytic: attacking group inorganic phosphate, results in 3’-OH and 5’-PP

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)

RNases

Family RNases Prokaryotic RNases Characteristics Exonucleases 3’ 5’

RNR RNase II nonspecific processive, degrades only ssRNA, mRNA decay

RNase R

nonspecific processive, degrades ssRNA and dsRNA, mRNA decay

DEDD RNase D distributive, small RNA and stabile RNA processing

RNase T

Oligoribonuclease

specific for oligoribonucleotides

RBN

RNase BN/Z

distributive exonuclease 3’- 5’ and endonuclease, tRNA processing

PDX PNPase phosphorolytic processive, degradosome subunit, KH/S1 RNA BD domains, degrades ss/dsRNA

RNase PH

phosphorolytic distributive

Exonucleases 5’ 3’

*RNAse J1/J2

present in Bacillus subtilis, specific for 5’ monoP ssRNA, mRNA decay

Endonucleases

RNase III

dsRNA specific, rRNA, tRNA, mRNA processing, mRNA degradation

RNase E

degradosome subunit, mRNA decay; rRNA tRNA and RNaseP RNA processing

RNase G

similar to RNase E

RNase I

nonspecific, mRNA degradation

RNase H

specific for RNA:DNA hybrid

RNase P

tRNA 5’ end processing

RNase Z

tRNA 3’ end processing

Rae1/YacP

ribosome-dependent mRNA decay in Bacillus subtilis

*RNAse J1/J2

mRNA decay in Bacillus subtilis

MazF/EndoA toxin, mRNA degradation in stress conditions, sequence specific

RNAse M5

Mackie, Nat. Rev. Microbiol, 2012

Novel bacterial endonuclease Rae1 involved in ribosome-

dependent mRNA decay in Bacillus subtilis

Leroy et al, EMBO, 2017

Bacterial exo- and endo-nucleases

Huiet al, Annu Rev genet, 2015

PNPazy trimer Symmons et al, Structure, 2000

Degradosome - major complex involved in mRNA decay in bacteria, functions as dimer RNase E 5’-phosphate -dependent endoribonuclease, N-terminal nucleolytic domain, C-terminal protein binding domain, central RNA binding domain (BD)

PNPase phosphorolytic processive exonuclease 3’ - 5’, KH and S1 RNA BD

RhIB ATP-dependent helicase, DEAD box, stimulate degradation of structured RNA regions Enolase glycolytic enzyme

additional DnaK/GroEL chaperons , poliphosphate kinase, poly(A) polymerase, S1 ribosomal protein

PDX domain ^{RNA BD} _(KH) ^{RNA BD} _(S1)

PNPaza

NH₂ COOH

RNase E

Catalytic domain RNA BD

(RRD) C-terminal

Degradation of bacterial mRNAs

Symmons et al, TiBS, 2002 PNPaza

RhIB

Degradation of bacterial mRNAs

Huiet al, Annu Rev genet, 2015

RNAse E or Y

General endo-dependend pathway

3’ exo-dependend pathway

5’-dependent pathway

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.

Symmons et al, TiBS, 2002 Mohanty et al, Mol. Microbiol., 2004

PNPase PAP I Hfq

Degradation of bacterial mRNAs

Protein Function Characteristics Exonucleases 5’ 3’

Xrn1

cytoplasmic, mRNA degradation

Rat1

nuclear, pre-rRNA, sn/snoRNA, pre-mRNA processing and degradation Rrp17/Nol12 nuclear, pre-rRNA processing

Exosome 3’ 5’

multisubunit exo/endo complex subunits organized as in bacterial PNPazy Rrp44/Dis3 catalytic subunit Exo/PIN domains, distributive, hydrolytic Rrp4, Rrp40 pre-rRNA, sn/snoRNA processing, mRNA degradation

Rrp41-43, 45-46 participates in NMD, ARE-dependent, non-stop decay Mtr3, Ski4

Mtr4 nuclear helicase cofactor

Rrp6 / Rrp47p

exonuclease nuclear cofactor RNase D homolog /RNA binding

Ski2,3,7,8

cytoplasmic exosome cofactors DEAD box helicase, GTPase Other 3’ 5’ exonucleases

Rex1-4

3’-5’ exonucleases, rRNA, snoRNA, tRNA processing RNase D homolog

mtEXO 3’ 5’

mitochondrial degradosome RNA degradation in yeast

Suv3/ Dss1

helicase/ 3’-5’ exonuclease DExH box/ RNase II homolog

Deadenylation

Ccr4/NOT

major deadenylase complex (Ccr, Caf, Pop, Not proteins) Ccr4- Mg²⁺ dependent endonuclease

Pop2

deadenylation regulator, deadenylase activity RNase D homolog

Pan2p/Pan3

additional deadenylases (poliA tail length) RNase D homolog, poly(A) specific nuclease

PARN

mammalian deadenylase RNase D homolog, poly(A) specific nuclease

Endonucleases

RNase III

-Rnt1 pre-rRNA, sn/snoRNA processing, mRNA degradation dsRNA specific

-Dicer, Drosha siRNA/miRNA biogenesis, functions in RNAi PAZ, RNA BD, RNase III domains

Ago2 Slicer

mRNA cleavage in RNAi

Utp24

early 35S pre-rRNA processing PIN domain

Nob1

18S pre-rRNA processing PIN domain

SMG6

mRNA cleavage in NMD PIN domain

RNase P

5’ tRNA end processing RNP complex

RNase MRP

pre-rRNA processing RNP complex, similar to RNase P

RNase L

rRNA degradation in apoptosis oligo 2-5A dependent (ppp(A2’p)nA)

ELAC2/Trz1

3’ tRNA endonuclease PDE motif and Zn^{2+ -}binding motif

Eukaryotic RNases

Protein Function / Characteristics 5’ 3’ decay: decapping

Dcp1/Dcp2

Dcp2- pyrophosphatase catalytic activity, Nudix domain, Dcp1- protein binding

Hedls/Ge-1/Edc4

decapping cofactor, WD40 domain

Edc1,2,3

decapping enhancers, stimulate cap binding/catalysis, Edc1-2 (yeast), Edc3 (all eykaryotes)

Dhh1

DexD/H ATPase, decapping activator by translation repression

Lsm1-7

decapping activator, heptameric complex, binds mRNA 3’ end-U rich tracts

Pat1

decapping activator by translation repression

TRAMP complex: nuclear RNA surveillance, polyadenylation-dependent degradation

Trf4/Trf5

nuclear alternative poly(A) polymerases

Mtr4

DEAD box helicase

Air1/Air2

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

RNA binding

Sen1

RNA helicase

Eukaryotic auxiliary decay factors

RNases

Stoecklin and M. hlemann, BBA, 2013

Kilchert et al, Nat Rev Mol Cell biol, 2016

EXOSOME: 3’ 5’ decay machinery

• 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

_Dz^iembo

wski et al, Mol.Cell, 2008; Nature, 2008

Dis3

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, ARE-

dependent decay

Kastenmayer and Green, 2000, PNAS

Crystal structure of S. pombe Rat1/Rai1 complex

Xiang et al, 2009, Nature

XRN family: 5’ 3’ processive exonucleases

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

• 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

Wang et al. PNAS, 2002 She et al. Nat.Struct. Mol. Biol, 2004

Dcp1

Dcp2

Gu et al., M.Cell, 2004

DCP- DECAPPING ENZYMES

DcpS

• DcpS: HIT pyrophosphatase („histidine triad” on the C-terminus)

• catalyses the cleavage of m

⁷

GDP -> m

⁷

GMP + Pi remaining after decapping during mRNA 5’ decay

• cooperates with the exosome during mRNA 3’ decay (m

⁷

GpppX-oligoRNA -> m

⁷

GMP+ pp-oligoRNA)

• functions as an asymmetric dimer

Nudt proteins (22), Nudt16, Nudt3 with in vivo decapping activity in mammalian cells

• Dcp1/Dcp2 complex participates in mRNA 5’ decay

• catalyses the reaction m

⁷

GpppX-mRNA -> m

⁷

GDP + 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)

Sm motif

Involved in pre-mRNA splicing

• associates with U6 snRNA

• required for U6 RNA accumulation and U6 snRNP biogenesis

• 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)

nuclear

cytoplasmic

Achsel et al, EMBO J, 2001

LSM PROTEINS

EUKARYOTIC mRNA

AAAAAAAAAAAAA

m

⁷

GpppG AUG UAA

5’ ss 3’ ss 50-200 nts

INTRON

5’UTR 3’UTR

UTR- UnTranslated Region

AAAAAAAAAAAAA

AUG UAA

50-200 nts

5’UTR 3’UTR

NUCLEUS

CYTOPLASM

m

⁷

GpppG CBP20

CBP80

eIF3

eIF4E

eIF4G

EJC

SPLICEOSOME

PABP2

PABP1

eIF4G eIF4E

m

⁷

Gppp

UAA A A A A

A A A

AUG

Pab1p

mRNA t

_1/2

= few minutes to 2 hours (yeast) to >90 hours (mammals)

ORF

mRNA DEGRADATION in the CYTOPLASM

Coller and Parker, Annu. Rev. Biochem., 2004

P-BODY ASSEMBLY RNA DEGRADATION or storage?

DEADENYLATION

RELEASE OF RIBOSOMES the

RELEASE OF TRANSLATION FACTORS

^t

he

RECRUITMENT OF DECAY

FACTORS (decapping)

AAAAA…..AAAAA Pat1p

Pab1p m 7 Gppp

eIF4G eIF4E

NORMAL mRNA DECAY

IN THE CYTOPLASM

m 7 Gppp AAAAA…..AAAAA Pat1p

dissociation

deadenylation deadenylacja Ccr4/Pop2/Not

Pan2/Pan3

NORMAL mRNA DECAY

IN THE CYTOPLASM

m 7 Gppp AAAA…AAA

Recruitment of Lsm/Dcp

Lsm1-7

Pat1p

NORMAL mRNA DECAY

IN THE CYTOPLASM

AAAA…AAA

Lsm

Pat1p

Egzosom

^C

Ski2p

Xrn1p

5’-3’degradation

3’-5’degradation

decapping

NORMAL mRNA DECAY

IN THE CYTOPLASM

• 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

TAIL-seq:

poly(A) tail analysis

TAIL-

In “strong translation mRNAs” PABP-eIF4G interaction stabilizes PABP binding to poly(A) allowing for poly(A) pruning to a defined length; “weak translation mRNAs”

are not protected by translation, poly(A) tails are shortened by deadenylases recruited to PABC domain, which triggers decapping and 5’-3’ decay

newly transcribed mRNAs

long poly(A) tails > 200nt

Balagopal and Parker, Cur.Op.Cel.Biol., 2009

mRNA DEGRADATION on POLYSOMES

mRNA DEGRADATION in P-BODIES

mRNA DEGRADATION in the CYTOPLASM

P bodies- processing bodies

P- bodies:

• cytoplasmic dynamic structures of mRNA and decay factors storage

(LSM, DCP, XRN, GW182)

• sites of mRNA degradation

(decay bodies, DCP bodies, GW bodies)

SPECIES:

Yeast

Human cells Drosophila C. elegans

CONTENT:

Dcp1/2 Lsm Edc1/2/3 Dhh1, Pat1 SMG5-7 GW182

AIN-1, ALG-1

general

human C. elegans miRISC

Xrn1 siRNA

hLSM1-GFP  -hDCP1 overlay

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

prt1ts prt1ts

Dcp2-GFP

23C 37C

Inhibition of translation activates P-bodies

Franks and Lykke-Andersen, 2008, Mol.Cell

Mutations in mRNA decay affect size and number of P-bodies

yeast

Sheth and Parker, 2003, Science;

Brengues et al, 2005, Science

Stress increases P-bodies

(aging, glucose deprivation, osmotic stress)

-GLU Dcp2-GFP

Dcp2-GFP

15 min 1M KCl

Hubstenberger et al, Mol Cell, 2017

• Purified PB contain mRNA regulons:

translationally repressed mRNAs with their regulatory proteins

• mRNAs with low protein yield are targeted to P-bodies

• mRNAs in PB are translationally repressed but not decayed

P-bodies:

mRNA decay or storage?

mRNA STABILITY

Elements in cis:

Chen and Shyu, TiBS 2016

LINK: mRNA DECAY and TRANSCRIPTION

Coupling between transcription and mRNA decay

Haimovich et al, BBA 2013

Dahan and Choder, BBA 2013

Coupling between transcription and mRNA decay

Transcriptional machinery regulates mRNA translation and decay in the cytoplasm

- PolII and promoters regulate cytoplasmic post-transcriptional stages

- Rpb4/7 subunits of PolII regulates trx initiation, elongation and polyadenylation by binding to the emerging transcript and remaining associated throughout its lifecycle:

(i) mRNA export; (ii) translation initiation via interaction with eIF3; (iii) deadenylation

and decay by Xrn1 and exosome via interaction with Pat1/Lsm1-7 complex

Haimovich et al, BBA 2013

Coupling between transcription and mRNA decay

Perez-Ortin et al, 2013, JMB

mRNA DECAY and TRANSCRIPTION mRNA homeostasis

Rpb4/7

Rpb4/7

Rpb4/7

Lsm1-7 Pat1

Kervestin and Jacobson, Nat Rev Mol Cel Biol,2012

mRNA SYNTHESIS and DECAY

RNA DECAY

PART II - SPECIFIC PATHWAYS

• 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)

RNA SURVEILLANCE =

RNA QUALITY CONTROL MECHANISMS

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

- degradation of mRNAs containing premature STOP codons (PTC) NMD

- 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

Amrani et al. Nat.Rev.Cel.Biol., 2006

Translation termination problem:

- premature termination or - ribosome stalling on PTC

mRNA degradation

NMD FACTORS

Behm-Ansmant et al., FEBS Lett, 2007 SMG7

Stalder and Muhlemann, TiCB, 2008

Llorka. Cur. Op. Chem. Biol. 2013

EJC contains proteins linked to different functions

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

Singh and Lykke-Andersen, TiBS 2003; Isken and Maquat, Gene Dev. 2007

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

active NMD complex Upf1-3 + SMG proteins

2. Assembly of the active NMD complex and repression of translation

EJC downstream of PTC is not removed by the advancing ribosome

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

MECHANISM OF NMD

MECHANISMS OF NMD

Kervestin Jacobson,

Nat Rev Mol Cel Biol,2012

EJC-dependent Alternative

long 3’UTR

Parker and Song, Nat. Struct. Mol. Biol. 2004; Isken and Maquat, Gene Dev. 2007

3. mRNA degradation

MECHANISM OF NMD

NMD 5’ 3’

deadenylation-independent decapping

exonucleolytic 5’ 3’ degradation (XRN1)

decapping is triggered by SMG7 recruitment or dephosphorylation of Upf1

XRN1 DCP1/2

exonucleolytic 3’ 5’

degradation

recruitment of the Exosome by Upf1

NMD 3’ 5’ _egzosom

^C

NMD endonucleolytic cleavage

(Drosophila melanogaster, humans)

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

Garneau et al, Nat. Rev. Mol. Cel. Biol. 2007

NGD

Dom34/Hbs1

Dom34 – has RNA-binding Sm fold Hbs1- GTPase binding activity

strong RNA structure

stalled translation

endonucleolytic cleavage (?)

Exosome

^C

Xrn1

NSD

Xrn1

ribosome stalls on poly(A) tail

Ski7

recruits the exosome

Exosome

^C

alternative pathway

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

NMD, NGD and NSD

Inada, TiBS 2016

AMD ARE - mediated decay

Roretz and Galouzi, J. Cell. Biol. 2008

TTP BRF1 HuR AUF1 KSRP

ARE-binding proteins (AUBP)

• Exosome (RRP45, RRP41, RRP43) is recruited by ARE-binding proteins AUBP (AUF1)

• Exosomal subunits interact directly with ARE sequences

• PARN and CCR4, deadenylases and XRN1, DCP1/2 interact with AUBP (TTP, KSRP, BRF1) mRNA instability elements:

- U rich - AU rich

AUUUA, UUAUUUA(U/A)

_n

- oligo(U) (or CUCU) tails - DST (plants)

GGAnnAUAGAUUnnnCAUUnnGUAU

• AMD degradation of mRNAs containing AU-rich instability elements

present in mRNA 3’ UTR

AMD - connections with RNAi?

The RNAi connection?

• 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

Georg Stoecklin’s website

Simms and Zaher, CellMolLifeSci 2016

RNA REPAIR

STAUFEN-mediated DECAY (SMD)

STAU1, a dsRNA binding protein, recruits UPF1 to target mRNA 3'UTRs to elicit SMD in a translation-dependent fashion.

SMD targets contain a STAU1 binding site (SBS) within their 3' UTR.

They include newly synthesized CBC-bound mRNAs and steady-state eIF4E-bound mRNAs.

Gong and Maquat, Nature 2011

Some mRNAs are targeted to SMD by lncRNAs

(Alu elements) which form SBS with SMD substrates.

Isken and Maquat, Nat Rev Genet 2008

OTHER MECHANISMS

Garneau et al, Nat. Rev. Mol. Cel. Biol. 2007

Endonuclease mediated decay

• 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

Exosome

^C

Xrn1

RNase MPR, RNase III (Rnt1)

IRE1, PMR1

Deadenylation-independent decay

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 (RPS28B and EDC1 mRNAs)

Garneau et al, Nat. Rev. Mol. Cel. Biol. 2007

OTHER MECHANISMS

Xrn1

RPS28B

Dcp1 Dcp2

Edc3 Rps28B

Casolari and Silver, TiCB., 2004

RES complex (REtention and Splicing): Snu17/Bud13/Pml1 Processing Quality Control Export

• nuclear retention of intron-containing pre-mRNAs

mRNA DECAY in the NUCLEUS

AAAA…AAA

AAAAAAA…AAAAAA m7Gppp

Lsm2-8

• pre- mRNA with unspliced introns

Exosome

^N

Mtr4p

m 7 Gppp m 7 Gppp

Rat1p

mRNA DECAY in the NUCLEUS

AAAA…AAA

m⁷G AAAAAA

NUCLEUS

CYTOPLASM

mRNA export blocked

Rat1p Lsm2-8

Dcp1/2p

m⁷G

AAAAAA

Rrp6

• mRNA arrested in the nucleus

mRNA DECAY in the NUCLEUS

TRAMP - EXOSOME COFACTOR (yeast)

YEAST

TRAMP = Trf4/5 + Air1/2 + Mtr4

polyadenylation

complex poly(A)

polymerases RNA binding

proteins RNA DEVH helicase

Polyadenylation-mediated nuclear discard pathway for defective RNAs

LaCava et al., Cell, 2005; Vanacova et al., PLoS Biol. 2005; Wyers et al., Cell, 2005; Lubas et al. Mol. Cell, 2011

Interacts with

- exosome via Mtr4 - Nrd1/Nab3 complex

some RNAs degraded by Rat1/Xrn1

• hypomodified tRNAs, pre-tRNAs

• ncRNAs:

sn/snoRNAs, rRNAs, some mRNAs

CUTs (Cryptic Unstable Transcripts)

Vanacova and Stefl, Embo Rep, 2007

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)

TRAMP/EXOSOME

Houseley et al., Nat. Rev. Mol. Cel. Biol. , 2006 Tuttuci and Stutz., Nat. Rev. Mol. Cel. Biol. , 2011

NEXT and PAXT - EXOSOME COFACTORS (humans)

Lubas et al. Mol. Cell, 2011; Meola et al., . Mol. Cell, 2016

• ZFC3H1 (Zn-knuckle protein) links MTR4 with PABPN1 in PAXT

• ZFC3H1/PABPN1 and RBM7/ZCCHC8 (NEXT) interact with MTR4 in a mutually exclusive manner

• PAXT and NEXT direct distinct RNA species for nuclear exosome degradation

• PAXT targets tend to be longer and more extensively polyadenylated than NEXT targets

HUMAN

ZCCHC8 Zn-knucle RMB7

RNA binding

NEXT

Nuclear Exosome

Targetting

Tutucci 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

S. cerevisiae Rai1 – Rat1 activator

5’-ppp pyrophosphohydrolase and phoshodiesterase-decapping nuclease (unmethylated cap-specific)

CAP NUCLEAR RNA SURVEILLANCE 5’-3’

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

Phizicky and Hopper , GeneGev.,2010

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)

tRNA SURVEILLANCE

pre-tRNA

(hypo-modified)

mature tRNA (hypo-modified)

tRNA SURVEILLANCE

Interplay between tRNA synthesis and degradation

Wichtowska, Turowski, Boguta, WIREsRNA, 2013

• 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

pre-tRNAs are DEGRADED by the EXOSOME

Dis3 exo Dis3

endo

Important contribution of the endo Dis3 activity to the degradation

of structured substrates

Lafontaine, TiBS.,2010

Nucleus:

pre-rRNAs

Nrd1/Nab3

(via pre-mature termination)

rRNA SURVEILLANCE

Cytoplasm:

mature ribosomes

NRD - nonfunctional rRNA decay

mutations in peptidyl transferase centre

mutations in decoding site

Lafontaine, TiBS.,2010

rRNA SURVEILLANCE

Dom34::Hbs1

factors involved in NGD and NSD Mms1, Rtt101-

subunits of E3 ubiquitin ligase complex

RNA SURVEILLANCE

pre-tRNAs

nucleus cytoplasm

nucleus

nucleus cytoplasm

nucleus NRD Nonfunctional rRNA Decay

RNA SURVEILLANCE

Vohhodina et al., WIREsRNA’16

Stoecklin and Mühlemann, BBA, 2013

SURVEILLANCE RNA

OLIGO-URIDYLATION

PUP Poly(U) Polymerases

TUTase Terminal Uridylyl Transferase 3’ oligouridylation 1. Histone mRNA degradation (metazoans)

Mullen and Marzluff, Genes Dev., 2008

3’hExo

Eri-1

3’hExo Eri-1

3’hExo Eri-1 Lsm1-7

OLIGO-URIDYLATION

PUP Poly(U) Polymerases

TUTase Terminal Uridylyl Transferase 3’ oligouridylation 1. Histone mRNA degradation (metazoans)

Mullen and Marzluff, Genes Dev., 2008

3’hExo

Eri-1

3’hExo Eri-1

3’hExo Eri-1

Scheer et al, TiG., 2016

1. Histone mRNA degradation (metazoans)

histone mRNA synthesis

histone mRNA decay

2. miRNA degradation

precursors C. elegans mammals

Krol et al., Nat Rev Genet, 2010; Kim et al., Cell, 2010

OLIGO-URIDYLATION

mature

Arabidopsis

Chlamydomonas

3. mRNA degradation? (plants)

Lsm1-7

DIS3L2

DIS3L2

hDIS3L2 - EXOSOME independent DECAY

pre-miRNA degradation

3’-5’ cytoplasmic mRNA decay

3’ degradation of aberrant, structured ncRNAs:

tRNA, sn/snoRNA, rRNA, lncRNA, Y RNA, vault RNA,

surveillance of 3’ snRNA processing

Krol et al., Nat Rev Genet, 2010

Pirouz et al., Cell Rep, 2016;

Ustianenko et al., EMBO, 2016

Thompson and Parker, Cell, 2009

(S. cerevisiae, D. melanogaster, A. thaliana, A. nidulans, human cell lines)

STRESS-INDUCED ENZYMATIC

tRNA and rRNA DEGRADATION

RNA DECAY

• 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

TAKE-HOME MESSAGE

Processing and degradation are often carried out by the same machineries

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

TAKE-HOME MESSAGE