RNA DECAY PART I - GENERAL MECHANISMS PART II - SPECIFIC PATHWAYS

Transcription

1 RNA DECAY PART I - GENERAL MECHANISMS PART II - SPECIFIC PATHWAYS

2 REGULATION OF GENE EXPRESSION 1) PolII assembly 2) transcription 3) mrna processing 4) mrna export 5) translation 6) protein stability 7) RNA degradation

3 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 H 2 O, 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

4 Prokaryotic RNases Family 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 RNase E RNase G RNase I RNase H RNase P RNase Z Rae1/YacP *RNAse J1/J2 MazF/EndoA RNAse M5 dsrna specific, rrna, trna, mrna processing, mrna degradation degradosome subunit, mrna decay; rrna trna and RNaseP RNA processing similar to RNase E nonspecific, mrna degradation specific for RNA:DNA hybrid trna 5 end processing trna 3 end processing ribosome-dependent mrna decay in Bacillus subtilis mrna decay in Bacillus subtilis toxin, mrna degradation in stress conditions, sequence specific

5 Mackie, Nat. Rev. Microbiol, 2012

6 Bacterial exo- and endo-nucleases Huiet al, Annu Rev genet, 2015 Novel bacterial endonuclease Rae1 involved in ribosomedependent mrna decay in Bacillus subtilis Leroy et al, EMBO, 2017

7 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 RNase E NH 2 Catalytic domain RNA BD (RRD) C-terminal COOH PNPaza PDX domain RNA BD (KH) RNA BD (S1) RhIB PNPaza PNPazy trimer Symmons et al, Structure, 2000 Symmons et al, TiBS, 2002

8 Degradation of bacterial mrnas General endo-dependend pathway 3 exo-dependend pathway RNAse E or Y 5 -dependent pathway Huiet al, Annu Rev genet, 2015

9 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. PNPase Hfq PAP I Mohanty et al, Mol. Microbiol., 2004 Symmons et al, TiBS, 2002

10 Protein Function Characteristics Exonucleases 5 3 Xrn1 Rat1 Rrp17/Nol12 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 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, 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 Rex 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- Mg 2+ 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) n A) ELAC2/Trz1 3 trna endonuclease PDE motif and Zn 2+ - binding motif

11 Eukaryotic auxiliary decay factors 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

12 RNases Stoecklin and M. hlemann, BBA, 2013

13 EXOSOME: 3 5 decay machinery Kilchert et al, Nat Rev Mol Cell biol, 2016 Dis3 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 Dziembowski et al, Mol.Cell, 2008; Nature, 2008

14 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

15 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) Xiang et al, 2009, Nature 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

16 She et al. Nat.Struct. Mol. Biol, 2004 Dcp2 DCP- DECAPPING ENZYMES Dcp1 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) Wang et al. PNAS, 2002 Nudt proteins (22), Nudt16, Nudt3 with in vivo decapping activity in mammalian cells 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) Gu et al., M.Cell, 2004 functions as an asymmetric dimer

17 LSM PROTEINS Sm motif Involved in pre-mrna splicing associates with U6 snrna Achsel et al, EMBO J, 2001 required for U6 RNA accumulation and U6 snrnp biogenesis nuclear interacts with the U4/U6.U5 tri-snrnp cytoplasmic Functions in mrna decapping and decay activator of decapping interacts with components of the mrna decapping and degradation machinery (XRN, DCP, Pat1)

18 EUKARYOTIC mrna CBP80 NUCLEUS SPLICEOSOME 5 UTR INTRON 3 UTR m 7 GpppG AUG UAA 5 ss 3 ss CBP20 PABP2 AAAAAAAAAAAAA nts CYTOPLASM eif3 EJC PABP1 5 UTR 3 UTR m 7 GpppG eif4e eif4g AUG ORF UAA AAAAAAAAAAAAA nts m 7 Gppp eif4e eif4g AUG A A Pab1p A A A A A UAA mrna t 1/2 = few minutes to 2 hours (yeast) to >90 hours (mammals) UTR- UnTranslated Region

19 mrna DEGRADATION in the CYTOPLASM DEADENYLATION RELEASE OF RIBOSOMES the RELEASE OF TRANSLATION FACTORS t he RECRUITMENT OF DECAY FACTORS (decapping) P-BODY ASSEMBLY RNA DEGRADATION or storage? Coller and Parker, Annu. Rev. Biochem., 2004

20 NORMAL mrna DECAY IN THE CYTOPLASM eif4g eif4e m 7 Gppp Pab1p AAAAA..AAAAA Pat1p

21 NORMAL mrna DECAY IN THE CYTOPLASM dissociation m 7 Gppp Pat1p AAAAA..AAAAA Ccr4/Pop2/Not Pan2/Pan3 deadenylacja deadenylation

22 NORMAL mrna DECAY IN THE CYTOPLASM Recruitment of Lsm1-7 Lsm/Dcp m 7 Gppp AAAA AAA Pat1p

23 NORMAL mrna DECAY IN THE CYTOPLASM Xrn1p 5-3 degradation decapping Lsm Pat1p Ski2p Egzosom C AAAA AAA 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

24 TAIL-seq: poly(a) tail analysis newly transcribed mrnas long poly(a) tails > 200nt 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

25 mrna DEGRADATION in the CYTOPLASM mrna DEGRADATION in P-BODIES mrna DEGRADATION on POLYSOMES Balagopal and Parker, Cur.Op.Cel.Biol., 2009

26 P- bodies: P bodies- processing bodies (decay bodies, DCP bodies, GW 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 general Xrn1 sirna SMG5-7 GW182 AIN-1, ALG-1 human C. elegans mirisc Cougot et al, JCB, 2004 mrna decay factors co-localize and polya + RNA accumulates in P bodies degradation bodies differ from stress granules

27 Mutations in mrna decay affect size and number of P-bodies Assembly of P bodies Stress increases P-bodies (aging, glucose deprivation, osmotic stress) Dcp2-GFP Dcp2-GFP -GLU Inhibition of translation activates P-bodies Dcp2-GFP prt1ts yeast prt1ts 23C 37C 15 min 1M KCl Sheth and Parker, 2003, Science; Brengues et al, 2005, Science Franks and Lykke-Andersen, 2008, Mol.Cell

28 P-bodies: mrna decay or storage? 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 Hubstenberger et al, Mol Cell, 2017

29 mrna STABILITY Elements in cis: Chen and Shyu, TiBS 2016

30 LINK: mrna DECAY and TRANSCRIPTION

31 Coupling between transcription and mrna decay Haimovich et al, BBA 2013

32 Coupling between transcription and mrna decay Dahan and Choder, BBA 2013 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

33 Coupling between transcription and mrna decay Haimovich et al, BBA 2013

34 mrna DECAY and TRANSCRIPTION mrna homeostasis Rpb4/7 Rpb4/7 Rpb4/7 Lsm1-7 Pat1 Perez-Ortin et al, 2013, JMB

35 mrna SYNTHESIS and DECAY Kervestin and Jacobson, Nat Rev Mol Cel Biol,2012

36 RNA DECAY PART II - SPECIFIC PATHWAYS

37 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)

38 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

39 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

40 NMD FACTORS Stalder and Muhlemann, TiCB, 2008 Llorka. Cur. Op. Chem. Biol SMG7 Behm-Ansmant et al., FEBS Lett, 2007

41 metazoan EXON JUNCTION COMPLEX (EJC) 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

42 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 active NMD complex Upf1-3 + SMG proteins 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 Singh and Lykke-Andersen, TiBS 2003; Isken and Maquat, Gene Dev. 2007

43 MECHANISMS OF NMD EJC-dependent Alternative long 3 UTR Kervestin Jacobson, Nat Rev Mol Cel Biol,2012

44 MECHANISM OF NMD 3. mrna degradation DCP1/2 XRN1 NMD 5 3 deadenylation-independent decapping exonucleolytic 5 3 degradation (XRN1) NMD endonucleolytic cleavage (Drosophila melanogaster, humans) SMG6 decapping is triggered by SMG7 recruitment or dephosphorylation of Upf1 NMD 3 5 egzosom C recruitment of the Exosome by Upf1 exonucleolytic 3 5 degradation Parker and Song, Nat. Struct. Mol. Biol. 2004; Isken and Maquat, Gene Dev. 2007

45 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 strong RNA structure NSD Dom34/Hbs1 stalled translation endonucleolytic cleavage (?) ribosome stalls on poly(a) tail Ski7 recruits the exosome alternative pathway ExosomeC Xrn1 Exosome C Dom34 has RNA-binding Sm fold Hbs1- GTPase binding activity Xrn1 Garneau et al, Nat. Rev. Mol. Cel. Biol. 2007

46 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

47 Inada, TiBS 2016 NMD, NGD and NSD

48 AMD ARE - mediated decay AMD degradation of mrnas containing AU-rich instability elements present in mrna 3 UTR ARE-binding proteins (AUBP) TTP BRF1 HuR AUF1 KSRP 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) Exosomal subunits interact directly with ARE sequences PARN and CCR4, deadenylases and XRN1, DCP1/2 interact with AUBP (TTP, KSRP, BRF1) Roretz and Galouzi, J. Cell. Biol. 2008

49 AMD - connections with RNAi? The RNAi connection? TTP binds Ago2 Georg Stoecklin s website 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

50 RNA REPAIR Simms and Zaher, CellMolLifeSci 2016

51 STAUFEN-mediated DECAY (SMD) Isken and Maquat, Nat Rev Genet 2008 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. Some mrnas are targeted to SMD by lncrnas (Alu elements) which form SBS with SMD substrates. Gong and Maquat, Nature 2011

52 OTHER MECHANISMS Endonuclease mediated decay RNase MPR, RNase III (Rnt1) IRE1, PMR1 Exosome C 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

53 Deadenylation-independent decay (RPS28B and EDC1 mrnas) OTHER MECHANISMS 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

54 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

55 mrna DECAY in the NUCLEUS pre-mrna with unspliced introns AAAA AAA m 7 Rat1p m 7 Gppp Gppp 7 Gppp Exosome N AAAAAAA AAAAAA Lsm2-8 Mtr4p

56 mrna DECAY in the NUCLEUS NUCLEUS mrna arrested in the nucleus Dcp1/2p Rat1p m 7 G Lsm2-8 mrna export blocked AAAA AAA AAAAAA Rrp6 m 7 G AAAAAA CYTOPLASM

57 TRAMP - EXOSOME COFACTOR (yeast) TRAMP = Trf4/5 + Air1/2 + Mtr4 polyadenylation complex poly(a) polymerases RNA binding proteins RNA DEVH helicase YEAST some RNAs degraded by Rat1/Xrn1 Polyadenylation-mediated nuclear discard pathway for defective RNAs hypomodified trnas, pre-trnas ncrnas: sn/snornas, rrnas, some mrnas CUTs (Cryptic Unstable Transcripts) Interacts with - exosome via Mtr4 - Nrd1/Nab3 complex LaCava et al., Cell, 2005; Vanacova et al., PLoS Biol. 2005; Wyers et al., Cell, 2005; Lubas et al. Mol. Cell, 2011

58 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

59 TRAMP/EXOSOME Houseley et al., Nat. Rev. Mol. Cel. Biol., 2006 Tuttuci and Stutz., Nat. Rev. Mol. Cel. Biol., 2011

60 NEXT and PAXT - EXOSOME COFACTORS (humans) Nuclear Exosome Targetting NEXT HUMAN ZCCHC8 Zn-knucle RMB7 RNA binding 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 Lubas et al. Mol. Cell, 2011; Meola et al.,. Mol. Cell, 2016

61 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

62 CAP NUCLEAR RNA SURVEILLANCE 5-3 S. cerevisiae Rai1 Rat1 activator 5 -ppp pyrophosphohydrolase and phoshodiesterase-decapping nuclease (unmethylated cap-specific)

63 CAP NUCLEAR RNA SURVEILLANCE 5-3 S. cerevisiae Dxo1: -phoshodiesterase -decapping nuclease exonuclease - no pyrophosphohydrolase Human DXO - 5 ppp pyrophosphohydrolase - phoshodiesterase -decapping nuclease exonuclease

64 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

65 trna SURVEILLANCE mature trna (hypo-modified) pre-trna (hypo-modified)

66 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

67 pre-trnas are DEGRADED by the EXOSOME Dis3 endo Dis3 exo Important contribution of the endo Dis3 activity to the degradation of structured substrates

68 rrna SURVEILLANCE Nucleus: pre-rrnas Nrd1/Nab3 (via pre-mature termination) Lafontaine, TiBS.,2010

69 NRD- nonfunctional rrna decay Cytoplasm: mature ribosomes rrna SURVEILLANCE mutations in peptidyl transferase centre mutations in decoding site Mms1, Rtt101- subunits of E3 ubiquitin ligase complex Dom34::Hbs1 factors involved in NGD and NSD Lafontaine, TiBS.,2010

70 RNA SURVEILLANCE pre-trnas cytoplasm nucleus nucleus NRD Nonfunctional rrna Decay nucleus cytoplasm nucleus

71 RNA SURVEILLANCE Vohhodina et al., WIREsRNA 16

72 RNA SURVEILLANCE Stoecklin and Mu hlemann, BBA, 2013

73 OLIGO-URIDYLATION PUP Poly(U) Polymerases TUTase Terminal Uridylyl Transferase 1. Histone mrna degradation (metazoans) 3 oligouridylation 3 hexo Eri-1 Lsm1-7 3 hexo Eri-1 3 hexo Eri-1 Mullen and Marzluff, Genes Dev., 2008

74 OLIGO-URIDYLATION PUP Poly(U) Polymerases TUTase Terminal Uridylyl Transferase 1. Histone mrna degradation (metazoans) 3 oligouridylation 3 hexo Eri-1 3 hexo Eri-1 3 hexo Eri-1 Mullen and Marzluff, Genes Dev., 2008

75 1. Histone mrna degradation (metazoans) histone mrna synthesis histone mrna decay Scheer et al, TiG., 2016

76 2. mirna degradation precursors C. elegans mammals OLIGO-URIDYLATION DIS3L2 mature 3. mrna degradation? (plants) Arabidopsis Chlamydomonas Lsm1-7 Krol et al., Nat Rev Genet, 2010; Kim et al., Cell, 2010

77 hdis3l2 - EXOSOME independent DECAY DIS3L2 pre-mirna degradation Krol et al., Nat Rev Genet, cytoplasmic mrna decay 3 degradation of aberrant, structured ncrnas: trna, sn/snorna, rrna, lncrna, Y RNA, vault RNA, surveillance of 3 snrna processing Pirouz et al., Cell Rep, 2016; Ustianenko et al., EMBO, 2016

78 STRESS-INDUCED ENZYMATIC trna and rrna DEGRADATION (S. cerevisiae, D. melanogaster, A. thaliana, A. nidulans, human cell lines) Thompson and Parker, Cell, 2009

79 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

80 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