RNA DEGRADATION AND QUALITY CONTROL RNA metabolism 21st November 2023 RNA degradation -general principles -RNA degradation machines RNA surveillance -noncoding RNAs -coding RNAs (mRNAs) LECTURE CONTENTS RNA metabolism 2023 (m)RNA Turnover: Why Should We Care? 1. Control of Gene Expression 2. Quality Control of RNA Biogenesis RNA metabolism 2023 RNA degradaDon in development Some examples RNA metabolism 2023 Wang et al. Nature Reviews Genetics 7, 185–199 (March 2006) | doi:10.1038/nrg1808 Gene expression during preimplantation embryo development RNA metabolism 2023 AAAAAAAAAnm7 G All steps of mRNA produc1on are controlled CYTOPLASM NUCLEUS IntronPromoter region TranscripNon iniNaNon Exon Gene Primary transcriptm7 G TRANSCRIPTION CAPPING Mature mRNAAAAAAAAAAnm7 G SPLICING POLYADENYLATION TRANSPORT TRANSLATION Ribosomes RNA metabolism 2023 Ribosomal RNA maturation is one of the most complex RNA linked processes in the cell and must be tightly controlled. RNA metabolism 2023 rRNA producDon is highly complex and energeDcally expensive RNA metabolism 2023 When the removal of decay products goes wrong RNA metabolism 2023 AVERAGE mRNA HALF LIFE RNA metabolism 2023 AVERAGE mRNA HALF LIFE E. coli: 4 min (2-10 min) Yeast: 22 min (4-40 min) Humans: 10 hours (0.5-24 hours) RNA degradaMon: → typical mRNAs in a somaMc cell last from minutes to hours and this is a funcMon of the balance between synthesis and degradaMon RNA metabolism 2023 • 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 mRNA stability and turnover RNA metabolism 2023 RNA degradaDon mechanisms RNA metabolism 2023 RNA is prone to nucleolysis 5’ 3’ 5’ 3’5’ 3’ ✂Endonuclease 5’ UTR coding sequence 3’ UTR RNA metabolism 2023 RNA degradation by nucleases RNA ENDONUCLEASE RNA metabolism 2023 RNaseA pankreatic endoribonuclease Monomer, 14 kDa Dimer Liu et al., PNAS 1998 Highly stable, Heat resistant Small – hard to remove RNA degradation by nucleases RNA metabolism 2023 RNA degradation by nucleases RNA ENDONUCLEASE cleaves 3’of RNase A C,U RNase T1 G RNase T2 GACU RNase U2 A in ssRNA RNase I GACU in ssRNA RNase III GACU in dsRNA RNase H RNA:DNA hybrid (oBen used to remove specific parts of RNA) RNA metabolism 2023 •DOI:10.1101/pdb.prot080788 RNAse protection assay RNA metabolism 2023 Ribosome footprin>ng analysis RNA metabolism 2023 3’ to 5’ RNA exonuclease 3’ to 5’ RNA exonuclease RNA metabolism 2023 RNA degrada5on by hydrolysis!! RNA metabolism 2023 mRNA DEGRADATION AAAm7GpppG AAAAAAAAA5’ 3’ 5’ 3’5’ 3’ Exonuclease Exosome Endonuclease The degradaLon must be closely regulated in order to prevent wholesale eliminaLon of all transcripts. ✂ Rat1, Xrn1 RNA metabolism 2023 RNA DECAY = Is mainly exonucleoly1c – RNAs can escape the decay by simply protec1ng 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 ter1ary structures Base modifica1ons mRNA stabilizing and destabilizing features m7GpppG PAB PAB PAB - Protein binding elements - RNA binding sequence elements - Structural elements AAAAAAAAAAAAA 5’UTR 3’UTR Protein and RNA binding Protein binding and secondary structures eIF4E Coding region RNA metabolism 2023 TranslaDon iniDaDon complex eIF-4G AUG AAU AAAAAAAAAAAAAAAAAA PAB PAB m7GpppG eIF-4A 4B eIF4E RNA metabolism 2023 AAAm7GpppG Deadenylases AAAAAAAAA ‘Normal’ mRNA degradaDon is iniDated by deadenylaDon eIF-4G AUG AAU AAAAA PAB PAB m7GpppG eIF-4A 4B eIF4E eIF-4G AUG AAU AAAAAAAAAAAAAAAAAA PAB PAB m7GpppG eIF-4A 4B eIF4E RNA metabolism 2023 Wilusz et al. Nat. Rev. Molec Cell Biol. 2:237; 2001 Deadenylation-dependent mRNA decay (in yeast) (Cap removal occurs when poly(A) tail ~10 nts.) (Dcp1/Dcp2) (Xrn1) RNA metabolism 2023 Molecular machines in mRNA degradation AAAm7GpppG AAAAAAAAA5’ 3’ 5’ 3’5’ 3’ Exonuclease Exosome Rat1, Xrn1 Monomer, very potent, highly processive needs cofactors and activation Endonuclease ✂ RNA metabolism 2023 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 exosome The RNA exosome and proteasome: common principles of degradaDon control RNA metabolism 2023 The RNA exosome and proteasome: common principles of degradation control RNA metabolism 2023 Bonneau et al., Cell, Oct. 2009 RNA is fed through the barrel RNA metabolism 2023 Wilusz et al. Nat. Rev. Molec Cell Biol. 2:237; 2001 Endoribonucleoly:c decay (Xrn1) exosome RNA metabolism 2023 Lim et al. Cell 2014 UridylaDon marks mRNA for decay RNA metabolism 2023 • The poly(A) tail is progressively shortened by a deadenylase enzyme unXl it reaches ~20 A residues or less • The PABPI becomes destabilized and weakening its interacXon with the 5’ cap and translaXon iniXaXon 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 suscepXble to 3’-5’ exonucleases •OligouridylaXon of short poly(A) tail recruits Lsm complex, which in turn recruits decaping aparatus and induces degradaXon at the 5’end • 5’ decapping and subsequent degradaXon (from the 5’ end) can occur independently of deadenylaXon DeadenylaDon dependent mRNA degradaDon RNA metabolism 2023 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)…… Regulatory sequence elements in mRNAs RNA metabolism 2023 Specialized mRNA turnover pathways - ARE-mediated mRNA decay RNA metabolism 2023 ARE-binding proteins affect mRNA stability, translaRon and subcellular localizaRon. • ARE’s (adenylate- uridylate-rich instability elements) in mRNA’s enhance deadenylaRon and decay rates (but some ARE’s can stabilize mRNA’s); • ARE’s are bound by factors (e.g., HuR/HuA, AUF1/hnRNP D) that modulate stability of AREcontaining mRNA’s; mechanism of acRon not clear Other elements found in the 5’ UTR and coding regions also modulate transcript stability. Regulatory sequences in mammalian mRNAs RNA metabolism 2023 ARE-Mediated mRNA Turnover - AU rich elements (50-150nts) AUUUA; UUAUUUA(U/A)(U/A); or U-rich - cis-acting element located in 3' UTRs of mRNAs - transcripts that encode proteins that require rapid changes in response to stimuli such changes in the cell cycle, growth factors, response to microorganisms, inflammatory stimuli, and environmental factors - 10% of mammalian mRNAs contain AREs - A diverse set of trans-acXng proteins bind to AREs. These proteins can mediate other protein interacXons that modulate mRNA stability. Various ARE-associated proteins can promote rapid mRNA turnover by promoXng enhanced decapping, deadenylaXon, exosome recruitment, endonucleolyXc cleavage or combinaXons of these. AlternaXvely, some proteins that bind to AREs can stabilize the mRNA. Goldstrohm & Wickens (2008) Nat Rev Mol Cell Biol 9:337 ARE RNA metabolism 2023 Example of ARE-mediated change in mRNA Levels before and aVer the DNA damage response Cevhar & Kleiman (2010) WIRE 1:193 Under non-damage conditions: - AUF1 competes with the PABP for poly(A) tail binding, exposing it to PARN; TTP (tristetraproline) and KSRP (KH-type splicing regulatory protein) recruit PARN and CCR4 to deadenylate prior to degradation by the exosome. Under DNA damage conditions: - Genes involved in the DNA damage response pathway are up-regulated. HuR is up-regulated and competes with AUF1 for binding to the same ARE region. Loss of AUF1 binding stabilizes PABP association with the poly(A) tail. HuR also competes with TTP and KRSP to prevent recruitment of the deadenylases and exosome. RNA metabolism 2023 RNA metabolism 2023 Extremely long lived mRNAs in humans? RNA metabolism 2023 RNA metabolism 2023 mRNA DEGRADATION mRNA DECAY QUALITY CONTROL RNA metabolism 2023 mRNA quality control RNA metabolism 2023 RNA quality control and degradaDon in the cytoplasm Aim: To prevent transla1on 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? RNA metabolism 2023 Alternative pre-mRNA splicing can create enormous diversity RNA metabolism 2023 RNA metabolism 2023 SchemaLc representaLon of alternaLve splicing (AS) paZerns inducing nonsense-mediated mRNA decay (NMD)-sensiLve mRNA isoforms Nogueira et al., 2021 RNA quality control in the cytoplasm: NMD Rehwinkel, Raes, Izaurralde, 2006 RNA metabolism 2023 NMD regulates the expression of transcripts associated with diverse cellular processes. Rehwinkel, Raes, Izaurralde, 2006 RNA metabolism 2023 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 2023 Nonsence muta1ons in the coding regions of mRNAs can lead to: 1. Genera1on of a premature termina1on codon (PTC) ® shorter protein product NONSENCE MEDIATED DECAY = NMD, SMD 2. Loss of the termina1on codon- nonstop message ® longer protein product NONSTOP DECAY = NSD Both these surveillance pathways depend on a ‘test’ round of translaRon that can detect the presence of stop codons. RNA quality control in the cytoplasm 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 - an ORF in their 5’ UTR 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) normal stoppremature stop cap AAAAAAAAAAAA last exon-exon juncXon >50-55 nt NMD = Nonsense-Mediated Decay RNA quality control in the cytoplasm: NMD RNA metabolism 2023 Nonsense-Mediated mRNA Decay - Specialized pathway that degrades mRNAs that contain premature translaRon terminaRon signals - Protects the cell from translaRng mRNAs that might produce truncated pepRdes that could lead to harmful dominant negaRve effects - Occurs in all eukaryotes. - 30% of disease-generaRng mutaRons result in premature stop codons - Up to 10-20% of the transcriptome is regulated by NMD - PTC-containing transcripts caused by point mutaRons, frameshiB mutaRons, mRNAs with faulty alternaRve splicing, pre-mRNAs that escape nuclear retenRon, mRNAs that contain upstream open reading frames, mRNAs that carry introns in 3´ untranslated regions, or mRNAs with long 3´ untranslated regions Czapllinski et al. (1999) Bioeassay 21:685 RNA metabolism 2023 NMD = Nonsense-Mediated Decay Two main steps: 1. PTC recogni1on 2. Ini1a1on of mRNA degrada1on RNA metabolism 2023 RNA metabolism 2023 Rules for eliciLng NMD according to the two models of NMD acLvaLon in human cells. RNA metabolism 2023 Examples of natural NMD substrates Lejeune, Biomedicines 2022 RNA metabolism 2023 Lejeune, Biomedicines 2022 Examples of natural NMD substrates (Conti and Izaurralde, 2005) PTC recogniDon Yeast Mammals DSE EJC SMD in mammals SMD = Staufen1 (Stau1) mediated decay. Independent of EJC. Doesn’t require splicing. Involves Stau1, Upf1. Upf1 Stau1 binds to the 3’ UTR of a subset of parRcular mRNAs Stau1 NMD versus Staufen1 decay pathways RNA metabolism 2023 Mechanism by which a stop codon is defined premature and the targeted mRNA is degraded differ across species: but in all depends on a test round of translaRon 1. Mammals -Cross-talk between terminaRng ribosome and downstream EJC (exon-juncRon complex) -Premature Stop Codons (PTC) defined as premature if they are located >50 nt upstream of an exonjuncRon complex - Mammals: PTC recogniRon relies on splicing or on Staufen protein (SMD) - depends on a ‘test’ round of translaRon that can detect the presence of stop codons. 2. Yeast and invertebrates - PTC recogniRon doesn’t depend on splicing, intronless mRNAs subjected to NMD - depends on loosely defined sequence elements, and/or on the interacRon with other proteins bound downstream on the mRNA - stop codon has to be in the “appropriate context” PTC recogniDon RNA metabolism 2023 Decay of NMD targets Yeast and mammals Drosophila NMD substrates are targeted for degradation via interaction with Upf proteins NMD Factors Associate With the EJC core NMD components 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 transla1on RNA metabolism 2023 RNA metabolism 2023 Nogueira et al., 2021 Simplified representaLon of the nonsense-mediated mRNA decay (NMD) model in mammalian cells Major role of 3 conserved proteins UPF1,2,3 (Up-Frameshift): UPF3 - nuclear protein, associates with EJC of spliced mRNAs (positioned 20-24 nt upstream of exon-exon boundary) has an RBD that contacts UPF2 UPF2 - perinuclear, associates with Upf3, UPF2/3 dimer binds mRNA via UPF2 surface, it can also interact with UPF1 UPF1 - cytoplasmic, RNA helicase, key component of NMD associates with eRF1 and eRF3 binds to Upf2/3 dimer regulated by phosphorylation in multicellular organisms - role for SMG1-7 proteins Recognition of PTC recruits UPF1 binding that can then interact with downstream UPF2/3 forming surveillance complex IniLaLon of mRNA degradaLon RNA metabolism 2023 NMD effectors and phenotypes across species Organism Effectors Phenotype Yeast: S. cerevisiae Upf1 Not essential Upf2 (Nmd2) Upf3 Worm: C. elegans SMG-2 (UPF1) Viable worms with SMG-3 (UPF2) morphological effects on genitalia SMG-4 (UPF3) SMG-1 SMG-5 SMG-6 SMG-7 Fruitfly: D. melanogaster UPF1 Required for cell-cycle UPF2 progression and proliferation UPF3 SMG1 SMG5 SMG6 Mammals: Mus musculus Upf1 (Rent1) Upf1 KO: embryonic lethal; Upf2 required for cell cycle progression Upf3 Upf2 KD: no effect on cell Smg1 viability Smg5 Smg6 Smg7 Rehwinkel, Raes, Izaurralde, 2006 RNA metabolism 2023 RNA metabolism 2023 Nonsense-mediated RNA decay and its bipolar function in cancer Nogueira et al., 2021 RNA metabolism 2023 Summary of nonsense-mediated mRNA decay (NMD) inhibiLon/escaping and acLvaLon strategies for cancer treatment Nogueira et al., 2021 Nonstop decay Nonstop decay (ND) targets mRNAs lacking an in-frame terminaRon codon Occurs by different mechanism than NMD, but is also translaRon dependent ND requires the cytoplasmic exosome and the associated Ski complex Independent of the decapping and deadenylaRon factors Ski7 C-terminal domain binds to free A-site of ribosome, and recruites the exosome and the Ski complex AAAm7GpppG Exosome + Ski complex SKI2 SKI3 SKI8 AUG RNA quality control in the cytoplasm: ND RNA metabolism 2023 NoGo decay Doma & Parker, 2006 What happens when ribosomes are stalled on mRNAs due to mRNA defects, or defects in ribosomes? RNA quality control in the cytoplasm: NoGo RNA metabolism 2023 NoGo decay Clement & Lykke-Andersen, 2006 Doma & Parker, 2006 RNA metabolism 2023 Clement & Lykke-Andersen, 2006 Summary of mRNA surveillance pathways in the cytoplasm RNA metabolism 2023 Summary of mRNA Surveillance Pathways Doma & Parker (2007) Cell 131:660 RNA metabolism 2023 RNA quality control of noncoding RNAs RNA metabolism 2023 Telomeric and heterochromaRn transcripts pre-rRNA spacers CUTs Introns Aberrant intermediates TRAMP complex Exosome Exosome acXvaXon PolyadenylaXon Substrate recognition Degradation pre- mRNA 5’-capping splicing polyA Proteins pre-tRNA cleavage trimming base modification CCA addition TranslaNon pre-rRNA base modification cleavage trimming Ribosomes pre- snoRNA 5’- capping cleavage trimming base modification snoRNPs pre- snRNA 5’- capping cleavage trimming base modification snRNPs D N A pri- miRNA Capping, polyA Cleavage , Drosha Cleavage , Dicer RNAi Nuclear RNA quality control Nuclear RNA surveillance of noncoding RNAs RNA metabolism 2023 PolyadenylaDon mediates surveillance of ncRNAs in the nucleus TRAMP complex adds polyA RNA metabolism 2023 RNA tagging for degradaDon NUCLEUS CYTOPLASM TRAMP + DIS3-EXOSOME TUTase + DIS3L2 PolyadenylaXon-mediated RNA degradaXon UUUUUUUUTUTase? DIS3L2 Uridylation-mediated RNA degradation RNA metabolism 2023 RNA surveillance of noncoding RNAs in the cytoplasm RNA metabolism 2023 Bonneau, 2009, Lebreton 2008, Schaeffer 2009 Tomecki et al. EMBOJ 2010, Staals et al., EMBOJ 2010 NUCLEUS CYTOPLASM The domain organization of DIS3 proteins RNA metabolism 2023 DIS3L2 degrades uridylated RNAs UsXanenko et al., 2013 RNA metabolism 2023 CLIP-seq approach to idenDfy DIS3L2 targets In vivo crosslinking Faehnle et al.,Nature 2014 RNA metabolism 2023 The U-tail distribution on DIS3L2 bound RNAs C D 0% 5% 0 2 4 6 8 10 12 14 Tail length [nt] 0 2 4 6 8 10 12 14 Tail length [nt] Adaptor removal DIS3L2 MUT CLIP replicates Mapping 26 million out of 86 million mapped Trimming of U tails Shorted then 18nt are discarted (15 million total) Unmapped Mapping 3 million out of 42 million mapped Statistic Uridylated reads 4Us Selected 50nt regions with a minimum of 20 reads and 25% uridylation. (confidence 95%) Uridylated reads NonUridylated reads miscRNA miRNA tRNA rRNA 5S rRNA snRNA mRNA lincRNA RNA type Percentageofuridylatedreads(%) Distribution of uridylated reads for different RNA classes 25 20 15 10 5 0 UsXanenko et al., EMBO J 2016RNA metabolism 2023 Missprocessed ncRNAs and transcripts from pseudogenes are urydilated UsXanenko et al., EMBO J 2016RNA metabolism 2023 TDS = TUT-DIS3L2 sureillance of ncRNAs in the cytoplasm Ustianenko et al., EMBO J 2016 RNA metabolism 2023 1. General mRNA turnover pathways - 5’®3 & 3’®5’ - Deadenylases - Decapping complex - Xrn1, exosome, DcpS 2. Aberrant RNA turnover pathways - Premature stop codons: nonsense-mediated mRNA decay (NMD) - No stop codons: non-stop mRNA decay (NSD) - ElongaXon stall: no-go mRNA decay (NGD) - TranslaXon into the 3’ UTR: ribosome extension-mediated mRNA decay (REMD 3. Specialized mRNA turnover pathways - ARE-mediated mRNA turnover: AU-rich elements in the 3’ UTR are bound by proteins that modulate the stability of mRNAs in response to regulatory signals 4. RNA surveillance of noncoding RNAs - oligo(A) mediated RNA quality control in the nucleus - oligo(U) mediated RNA surveillance in the cytoplasm Lecture overview RNA metabolism 2023