LECTURE CONTENTS RNA degradation -general principles -RNA degradation machi RNA surveillance -coding RNAs (mRNAs) -noncoding RNAs m)RNA Turnover: Why Should We Care? Control of Gene Expression Quality Control of RNA Biogenesis AVERAGE mRNA HALF LIFE AVERAGE mRNA HALF LIFE E. coir. 4 min (2-10 min) Yeast: 22 min (4-40 min) Humans: 10 hours (0.5-24 hours) RNA degradation: —► typical mRNAs in a somatic cell last from minutes to hours and this is a function of the balance between synthesis and degradation mRNA stability and turnover • The concentration of mRNA is a function of both the rate of mRNA synthesis and the rate of mRNA degradation • The stability of mRNA also determines how rapidly synthesis of the encoded protein can be shut down —► e.g. for a stable mRNA, protein synthesis can persist long after transcription of the gene is repressed —► mRNA half life of most multicellular eukaryotic cells is many hours (compared to just a few minutes for bacteria) • Some proteins in eukaryotic cells are required for very short periods of time and are expressed in bursts (e.g. many signaling molecules like cytokines or cell cycle regulated transcription factors, such as c-fos) • Regulating the stability of mRNA is one way of ensuring that proteins are present for only short bursts or for longer periods of time, as is needed Gene expression during preimplantation embryo development Unfertilised 1-celL 2-c ell 4-cell 8-cell Morula Blastocyst Wang etal. Nature Reviews Genetics!, 185-199 (March 2006) | doi:10.1038/nrg1808 RNA degradation mechanisms RNA is prone to nucleolysis 5' UTR coding sequence 3' UTR Endonuclease RNA degradation by nucleases : RNA ENDONUCLEASE ♦ ♦ «1 HO —fl = 0 i RNA degradation by nucleases Monomer, 14 kDa RNaseA pankreatic endoribonuclease Highly stable, Heat resistant Dimer Small - hard to remove Liu et al., PNAS 1998 RNA degradation by nucleases RNA ENDONUCLEASE Base (C or U) OH i HO-fJ =0 6 R NAse A z^0^ Base (C or U) 4- CH20h .0. base 0 Base 0 h 0 —f! =0 OH = 0 HO —1^ = 0 i OH RNase A RNase T1 RNase T2 RNase U2 RNase I RNase III RNase H (often used to remove specific parts of RNA) cleaves 3'of C,U G GACU A in ssRNA GACU in ssRNA GACU in dsRNA RNA:DNA hybrid mRNA DEGRADATION Rati, Xrn1 Exonuclease Endonuclease AAAAAAAAAAAA 3' 5' 3 33w Exosome The degradation must be closely regulated in order to prevent wholesale elimination of all transcripts. RNA DECAY Is mainly exonucleolytic - RNAs can escape the decay by mply protecting their ends with proteins and/or by structural elements. Examples: mRNA: 5' 7mGpppG cap plus cap-binding proteins 3' poly(A) tail with poly(A) binding proteins bound tRNA, rRNA, snRNA, snoRNA: complex secondary and tertiary structures Base modifications mRNA stabilizing and destabilizing features Protein binding elements RNA binding sequence elements Structural elements 7GpppG 5' UTR Coding region 3'UTR Protein binding and secondary structures Protein and RNA binding Translation initiation complex ÉNormaľ mRNA degradation is initiated by deadenylation AAAAAA Uridylation marks mRNA for decay Ribosome PABP Deadenylation m7G Uridylation I I m'G. ***A(n< -25). TUT4 or TUT7 Uridylation-dependent decay / \ 5-3' decay LSM1-7 i-i 3-5' decay Exosome Lim et al. Cell 2014 DCP1/2 ^) DIS3L2 Deadenylation dependent mRNA degradation • The poly(A) tail is progressively shortened by a deadenylase enzyme until it reaches -20 A residues or less • The PABPI becomes destabilized and weakening its interaction with the 5' cap and translation initiation factors and also leads to an exposed 5' cap •Some mRNAs are cleaved internally by endonucleases (e.g. the miRISC) before they are further degraded by 3' -5' exonucleases • 5' caps can then be removed by decapping enzymes and unprotected 5' end is degraded by 5' -3' exonucleases • The shortened poly(A) tail is also susceptible to 3' -5' exonucleases •Oligouridylation of short poly(A) tail recruits Lsm complex, which in turn recruits decaping aparatus and induces degradation at the 5'end • 5' decapping and subsequent degradation (from the 5' end) can occur independently of deadenylation Regulatory sequence elements in mRNAs Encoded: • AU rich elements (ARE) in 3' UTRs - binding of specific proteins that recruit the exosome • Iron-responsive element (IRE) and iron regulatory protein (IRP) • Cell cycle-regulated histone mRNA stem-loop determinant (SL/SLBP) • Cytoplasmic polyadenylation element (CPE)...... Translational control Subcellular localization Stability 5' UTR 3' UTR Molecular machines in mRNA degradation Exonuclease Exosome Rati, Xrn1 Monomer, very potent, highly processive needs cofactors and activation The exosome Associates with specific co-factors depending on localization 2 forms: nuclear and cytoplasmic Exosome is poorly active in vitro and needs cofactors for activation The RNAexosome and proteasome: common principles degradation control Nature Reviews | Molecular Cell Biology The RNA exosome and proteasome: common principles of degradation control a Core complexes bound to ATP-dependent regulators b Core complexes bound to ATP-independent regulators Nature Reviews | Molecular Cell Biology mRNA DEGRADATION mRNA DECAY QUALITY CONTROL mRNA quality control All steps of mRNA production are controlled Promoter region Transcription initiation eg ion ,—> «- Intron Exon Gene TRANSCRIPTION CAPPING m7G Primary transcript SPLICING POLYADENYLATION NUCLEUS m7G AAAAAAAAAn Mature mRNA TRANSPORT TRANSLATION m7G CYTOPLASM AAAAAAAAA, Ribosomes rRNA production is highly complex and energetically expensive h- ■ pre-5S rRNA -rDNA repeat (9.1 kb)-- 35S pre-rRNA pre-5S rRNA B ITS1 Primary RNA pol I transcript Pseucouridylation (T) 2'-0-ribose methyiation Base methyiation? ITS2 H/ACA-box snoRNPs C/D-box snoRNPs Primary RNA pol III transcript Rntlp RNA82? 35S Cleavage An Rn*1P? j U3snoRNP 33S Cleavage Ai 32S Cleavage A2 U3 U14 snRtO snR30 snoRNPs Dbp4p DbpSp Fall p Roklp Rrp3p Dimlp Rrp5p U3 U14 snR10 snR30 snoRNPs Dbp4p DbpSp Fahp Roklp Rrp3p Dimlp Rrp5p DbpGp Dbpdp Nop4p Nop8p Dbp7p Mak5p Dralp? Base methyiation? Processing B2 RNA82? Processing Bn_ 20S 7SS 1 Cbf5p Nop2p Spblp DbplOp Spb4p Processing Nip7p C( + C2 7S, Cbf5p Nop2p Spb1 p Dbp10pSpb4p Nip7p 5.8SS Exosome Doblp 25S Exonuclease E*-C2 5.8S. Exosome Doblp 25S 5S Ribosomal RNA maturation is one of the most complex RNA linked processes in the cell and must be tightly controlled. Undepleled U3 sooRNA-depleted Utp7-depleted •Y rDNA repeat unit (9.1 kb) terminator/ *•- promoter enhancer terminator/ -- promoter enhancer Bii Bi. *2 A,3N|f^ °2 C^ Primary Transcript Co-transcnptonal Cleavage in 3' ETS ■L r Rntlp 35S- Box C*D snoRNPs Box H+ACA snoRNPs Endonudease - Rntlp Endonudease - RNase MRP Dimethylase - Dim ip Exosome components RNA helicases 5-XT exonudeases Assembly factors 3\>5' exonudeases OSiers 2,-0-methylation Pseudouridine formation CH, „. . Methyiation guide snoRNAs (C+D) Noplp . Pseudouridine guide snoRNAs (H+ACA) Garlp Cbt5p Lhp2p NcplOp CH, 35S Cleavage Ao 33S Cleavage A, U3 Noplp Sottp MpptOp Nop58p Nop56p Imp2lmp3p I U3 UU Noplp Sottp MpplOp Nop58p Nop56p Imp2lmp3p snR30 snR 10 Garlp CbtSp Lhp2p Nop 10p Rrp3p Rok Ip Pal Ip Rrp5p LcpSp RrpTp Rnt Ip Dim Ip 60S Synthesis factors Drslp Sbp4p Dbp3p Dpb6p Nip7p Nop2p Nop3p NopAfUp 32S Cleavage A. U3 UU Noplp Sotlp MpplOp NopSSp NopS6p Imp2 Imp3p snR30 snR 10 Garlp CbtSp Lhp2p Nop 10p Rrp3p Roklp Fallp RrpSp LcpSp RrpTp Rntlp Dim Ip 20S Base Dimethlyabon Dimlp 27SA, m A 20S Cleavage D m{A 18S Cleavage A, 27SA3 • Exonuclease A3 •> B|S 27SBS Rrp4p Rrp40p Rrp4a> np43p Rtp4m npltji Rrp46p Mtr3p CstOp Rrpep Doblp MRP RNA Pop1pPop3p Pap4pPcpSp Processing Popep Popfp 82 Poap Rprip Snm Ip RrpSp -Rna82p — Ratlp Xmlp Processing Processing - RnaS2p 27SB 7SS Processing C. + C, Exonuclease C,->E 5.8Sc 25S Rrp4p Rrp40p Rrp41p Rrp42p Rrp43t Rrp45p Rrp46p Mtr3p Csl4p Rrp6p Doblp at 7S, Processing C, + C? Exonuclease C,->E 5.8SL 25S When the removal of decay products goes wrong mRNA quality control and degradation in the cytoplasm Aim: To prevent translation of mRNAs that would generate aberrant proteins -targets mainly mRNAs -Almost 20% of mRNAs in humans have a premature stop codon. All of these are degraded by NMD. Where do you think all these mistakes are coming from? That is, which process in the biogenesis of an mRNA molecule is the most prone to errors? Alternative pre-mRNA splicing can create enormous diversity A exons B exons C exons D exons DSCAM gene ii A8C16 mRNA B24 D2 one out of 38,016 possible splicing patterns Figure 7-89. Molecular Biology of the Cell, 4th Edition. RNA quality control in the cytoplasm: NMD z 3 % "as o *-> o 3 35% of alternative isoforms are NMD candidates 10000 £ 8000 o s •1 > s I— •1 60O0 £ 4000 2000 3704 3127 4A Alternative isoforms NMD-candidates Alternative isoforms Not NMD-candidates Canonical isoforms Rehwinkel, Raes, Izaurralde, 2006 NMD regulates the expression of transcripts associated with diverse cellular processes. r.-! :: 190 B ONA repair □ Development ■ Transcription □ Translation ■ Transport □ Mitochondrion □ Cytoske*cton organization | Strudural constrtuenl and biogenesis of ribosome □ Cel cycle □ Not annotated Rehwinkel, Raes, Izaurralde, 2006 RNA quality control in the cytoplasm: NMD NMD = Nonsense-Mediated Decay Is initiated when mRNA contains: - a premature stop codon - an in-frame stop codon within a retained intron - an extended 3' UTR due to improper polyadenylation site use -anORFintheir5' UTR premature stop normal stop AAAAAAAAAAAA t last exon-exon junction It has been estimated that 30% of inherited genetic disorders in humans result from nonsense mutations or frameshift mutations, which generate PTCs Yet, most of these diseases are recessive (i.e. the truncated protein is not made and thus cannot interfere with the function of the wild type protein) Nonsense-Mediated mRNA Decay Specialized pathway that degrades mRNAs that contain premature translation termination signals min after transcriptional arrest 0 3 6 9 12 18 min after transcriptional arrest 0 3 6 9 12 18 Czapllinski ef a/. (1999) 6/oeassay 21:685 PGK1 t, 2= 45 min PGK1 t,^= 3 min Protects the cell from translating mRNAs that might produce truncated peptides that could lead to harmful dominant negative effects Occurs in all eukaryotes. 30% of disease-generating mutations result in premature stop codons Up to 10-20% of the transcriptome is regulated by NMD PTC-containing transcripts caused by point mutations, frameshift mutations, mRNAs with faulty alternative splicing, pre-mRNAs that escape nuclear retention, mRNAs that contain upstream open reading frames, mRNAs that carry introns in 3' untranslated regions, or mRNAs with long 3' untranslated regions NMD = Nonsense-Mediated Decay Two main steps: 1. PTC recognition 2. Initiation of mRNA degradation PTC recognition Yeast m7G PTC, Hrplp AAAAAAA DSI m7G DSE Pablp AAAAAAA 3* UTR Mammals Mammals m7G PTC, EJC ^AAAAA EJC Exon-exon boundary m7G PTC Wb1p AAAAAAA 3* UTP 'Conf/ and Izaurralde, 2005) SMD in mammals SMD = Staufenl (Staul) mediated decay. Independent of EJC. Doesn't require splicing. Involves Staul, Upf1. m7G Staul AAAAAAA 3' UTR Staul binds to the 3' UTR of a subset of particular mRNAs Decay of NMD targets Yeast and mammals m7G Decapping XRN1 NMD complex MAAAAA Deadenylation Exosome ♦ Ski complex Drosophila m7G m7G> NMD complex il ::::: Endonucteolytic cleavage ------- Exosome XRN1 + Ski complex NMD substrates are targeted for degradation via interaction with Upf proteins NMD Factors Associate With the EJC EJC core Direct binding important for NMD Possible binding Involved in NMD Core NMD Components: UPF3: associates with the EJC in the nucleus UPF2: perinuclear and binds to Upf3 as the mRNA is exported UPF1: associates at the stop codons in mRNAs during translation Aberrant mRNA Decay Pathways A. Nonsense-mediated mRNA decay (NMD) - Degrades mRNAs with premature stop codons B. Nonstop mRNA decay (NSD) - Degrades mRNAs without a stop codon C. No-go mRNA decay (NGD) - Degrades mRNAs that have a stalled ribosome D. Ribosome extension-mediated decay (REMD) - Degrades mRNAs where ribosome translates past the stop codon and into the 3' UTR RNA metabolism 2020 RNA quality control of noncoding RNAs All steps of mRNA production are controlled Promoter region Transcription initiation eg ion ,—> «- Intron Exon Gene TRANSCRIPTION CAPPING m7G Primary transcript SPLICING POLYADENYLATION NUCLEUS m7G AAAAAAAAAn Mature mRNA TRANSPORT TRANSLATION m7G CYTOPLASM AAAAAAAAA, Ribosomes Ribosomal RNA maturation is one of the most complex RNA linked processes in the cell and must be tightly controlled. Undepleled U3 sooRNA-depleted Utp7-depleted •Y rDNA repeat unit (9.1 kb) terminator/ *•- promoter enhancer terminator/ -- promoter enhancer Bii Bi. *2 A,3N|f^ C? C^ Primary Transcript Co-transcnptonal Cleavage in 3' ETS r Rntlp 35S- Box C*D snoRNPs Box H+ACA snoRNPs Endonudease - Rntlp Endonudease - RNase MRP Dimetbylase - Dim ip Exosome components RNA helicases 5-XT exonudeases Assembly factors 3\>5' exonudeases OSiers 2,-0-methylation Pseudouridine formation CH, „. . Methylation guide snoRNAs (CtD) Noplp . Pseudouridine guide snoRNAs (H+ACA) Garlp Cbt5p Lhp2p NoplOp CH, 35S Cleavage Ao 33S Cleavage A, U3 Noplp Sotlp MpptOp Nop58p Nop56p Imp2lmp3p I U3 UU Noplp Sotlp MpplOp Nop58p Nop56p Imp2lmp3p snR30 snR 10 Garlp CbtSp Lhp2p Nop 10p Rrp3p Rok Ip Pal Ip Rrp5p LcpSp RrpTp Rnt Ip Dim Ip 60S Synthesis factors Drslp Sbp4p Dbp3p Dpb6p Nip7p Nop2p Nop3p NopATHp 32S Cleavage A. U3 UU Noplp Sotlp MpplOp NopS8p NopS6p Imp2 Imp3p snR30 snR 10 Garlp CbtSp Lhp2p Nop 10p Rrp3p Roklp Fallp RrpSp LcpSp RrpTp Rntlp Dim Ip 20S Base Dimethlyabon Dimlp 27SA, m A 20S Cleavage D m{A 18S Cleavage A, 27SA3 • Exonuclease Ao •> Bis 27SBS Rrp4p Rrp40p Rrp4a> np43p Rtp4m ftrp45p RrMCfa Mtr3p Csl4p Rrpep Doblp MRP RNA Pcp1pPop3p Pap4pPcpSp Processing Popep Popfp 82 Po8p Rprip Snm Ip RrpSp -Rna82p — Ratlp Xmlp Processing Processing - RnaS2p 27SB 7SS Processing C. + C, Exonuclease C,->E 5.8Sc 25S RrpAp Rrp40p Rrp41p Rrp42p Rrp43) Rrp45p Rrp46p Mtr3p Csl4p Rrp6p Doblp at 7S, Processing C, + C? Exonuclease C,->E 5.8SL 25S Nuclear RNA surveillance of noncoding RNAs TRAMP complex Polyadenylation mediates surveillance of ncRNAs in the nucleus TRAMP complex adds polyA Unwinding Activation of exosome Nuclear and cytoplasmic RNA surveillance TRAMP + DIS3-EXOSOME Vanacova et ai, 2005, La Cava et ai, 2005, Wyers et ai, 2005 Mammalian TErminal NucleoTidyltransferases cofactor TENT4A PAPD7/PolS/TUT5 TENT4B PAPD5/TRF4-2 TENT2 PAPD4/GLD-2 TENT6 PAPD1/TUT2/mtPAP TENT1 TUT1/STARPAP TUT4/ZCCHC11 TUT7/ZCCHC6 TENT5 A,B,C,D FAIV □wo- Qt-M— specificity A, G A, G A A U,A U U A J Catalytic domain 1 Inactivated catalytic domain | Central domain ■ RRM Zinc finger Zinc knuckle Mammalian TErminal NucleoTidyltransferases cofactor TENT4A PAPD7/PolS/TUT5 TENT4B PAPD5/TRF4-2 TENT2 PAPD4/GLD-2 TENT6 PAPD1/TUT2/mtPAP TENT1 TUT1/STARPAP TUT4/ZCCHC11 TUT7/ZCCHC6 TENT5 A,B,C,D FAIV NTD I-1 IB- specificity A, G A, G A A A U,A U U A J Catalytic domain 1 Inactivated catalytic domain | Central domain Q RRM Zinc finger Zinc knuckle Mixed A/G tailing by TENT4A/B stabilizes mRNAs ^ead counts Nucleotides 1,457,817 42,157 17,325 14,064 13,206 7,701 5,844 4,678 2,728 2,616 AAAAAAAAAA AAAAAAAAAG AAAAAAAAA AAAAAAAAA AAAAAAAAGA AAAAAAAACA AAAAAAAA A AAAAAAAGAA AAAAAAA AA AAAAAAA AA AAAAAG AAAAGA AAAGAA AAGAAA p AGAAAA J GAAAAA J "444 AAA 0.5 1.0 1.5 2.0 Frequency (%) Rapid tail shortening Mixed tailing "44AAAA, .AGAAv ) "4/\AAAA AG (n) TENT4A or TENT4B Delayed tail shortening * CNOT complex Narry Kim lab, Science 2018 Mixed A/G tailing by TENT4A/B stabilizes mRNAs fast RNA polymerase II transcription capping <\/\s^\/>J>A_i:tl l30AA polyadenylation tent4a/B mixed A/G tailing \ SO-ISO^1 ,AloA( ,AA deadenylation a jT\CNOT6L V \cnot7 slow deadenylation ^jf\cnot6l cVbarv^AA>2o.5oAA( 5-3' XRN iC^uvAA^^ 3 -5 exosome rapid degradation Terminal nucleotidyl transferases (TENTs) in mammalian RNA metabolism, Volume: 373, Issue: 1762, DOI: (10.1098/rstb.2018.0162) Mammalian TErminal NucleoTidyltransferases cofactor TENT4A PAPD7/POIS/TUT5 TENT4B PAPD5/TRF4-2 TENT2 PAPD4/GLD-2 TENT6 PAPD1/TUT2/mtPAP TENT1 TUT1/STARPAP TUT4/ZCCHC11 TUT7/ZCCHC6 TENT5 A,B,C,D FAIV NTD I-1 specificity A, G A, G A A U,A U U A Catalytic domain I Inactivated catalytic domain J Central domain B RRM Zinc finger Zinc knuckle Mammalian TENTs cofactor TENT4A PAPD7/PolS/TUT5 TENT4B PAPD5/TRF4-2 TENT2 PAPD4/GLD-2 TENT6 PAPD1/TUT2/mtPAP TENT1 TUT1/STARPAP TUT4/ZCCHC11 TUT7/ZCCHC6 TENT5 A,B,C,D FAIV NTD I-1 specificity A, G A, G A A U,A U U A Catalytic domain I Inactivated catalytic domain J Central domain Q RRM Zinc finger Zinc knuckle TUT4 & TUT7 The most extensively studied protein from the whole family Multiple diverse roles Multiple targets: mRNA, ncRNAs Act in both: RNA processing and degradation Essential for: Germline development Differentiation Viral infection Stress response Apoptosis Cancer progression Inhibition of retrotransposition TUT4/7 in noncoding RNA metabolism Uridylation of miRNA precursors can either stimulate processing or trigger RNA degradation. Embryonic stem cells or certain cancer cells Differentiated cells Pre-miRNA Lin28 Let-7 pre-miRNA Lin28 TUT4. TUT7 °^0U (Prolonged btnding) Processive oligo-uridylation 1 Degrade Group II pre-miRNA If TUT7. TUT4 ,TUT2 (Infrequent binding) Mono-uridylation ! Promote miRNA biogenesis Ago2 or unknown nuclease 3' trimmed pre-miRNA TUT7, TUT4. , \ (TUT2) (Frequent binding) Distributive oligo-uridylation 1 Degrade NarryKim, EM BO J 2014 The role of let-7 in differentiation Stem Cells Differentiation Differentiated Cells LIN28 Let-7 miRNAs Highly Aggressive Tumors Tissue Repair Indueed Pluripotent Stem Cells (iPSCs) Neural cells Transformation Healing Reprogramming T Normal Cells Injured tissue Differentiated Cells Blood cells Cardiac muscle Mammalian TENTs cofactor TENT4A PAPD7/PolS/TUT5 TENT4B PAPD5/TRF4-2 TENT2 PAPD4/GLD-2 TENT6 PAPD1/TUT2/mtPAP TENT1 TUT1/STARPAP TUT4/ZCCHC11 TUT7/ZCCHC6 TENT5 A,B,C,D FAIV NTD I-1 specificity A, G A, G A A U,A U U A Catalytic domain I Inactivated catalytic domain J Central domain Q RRM Zinc finger Zinc knuckle DIS3L2 targets uridylated precursors of let-7 miRNA DIS3L2 in cancer and dissease nature on. genetics 2012 Germline mutations in DIS3L2 cause the Perlman syndrome of overgrowth and Wilms tumor susceptibility Pmijn\ Farida Latif & Eamonn R Mahcr DIS3L2 oligo(U) specificity Ustianenko et al., 2013 Faehnle eŕ al., 2014 Perlman syndrome association with DIS3L2 mutation • (Astuti et ai, Nature Genetics, 2012) rare genetic disorder fetal overgrowth developmental delay kidney abnormalities high risk of bilateral tumors and Wilms' tumor - a rare kidney cancer poor prognosis, high neonatal mortality link between DIS3L2 dysfunction and a disease phenotype remains unknown DIS3L2 knock down in somatic cells results in cell cycle defects > 9) -Q O C s o E CD -Q 350 300 250 200 150 100 50 Control DIS3L2 K.D. Oligo 1 ■ Oligo 2 I Jl JÍ a® # <£' <í>v <->v <<# *ř J" *ř J" *f # ^ Metaphase error Telophase error Polylobed Anaphase errors Binucleated Protein