2024 MRes MMB8008 Chromosome Biology and Cell Cycle Control Quiz PDF

MMB8008 Chromosome Biology and Cell Cycle Control in Health and Disease The importance of RNA surveillance in eukaryotic gene expression Dr Claudia Schneider Newcastle University Biosciences Institute [email protected]...

MMB8008 Chromosome Biology and Cell Cycle Control in Health and Disease The importance of RNA surveillance in eukaryotic gene expression Dr Claudia Schneider Newcastle University Biosciences Institute [email protected]  Learning Outcomes Gain knowledge of different eukaryotic RNA surveillance pathways and how they are linked to genome maintenance Recap key factors and basic mechanisms in RNA degradation Consider what makes RNAs susceptible to surveillance, and how different substrates are targeted for degradation Get insights into the molecular mechanisms by which the RNA exosome distinguishes between stable and unstable RNAs Learn how defects in RNA surveillance can lead to genome instability and disease  Lecture organisation Part 1: Surveillance of protein-coding messenger RNAs Introduction into mRNA degradation and key enzymes Specialised mRNA quality control pathways Nonsense-mediated decay (NMD) and genome stability Part 2: Transcriptome-wide RNA surveillance and the RNA exosome The ncRNA world The RNA exosome, its co-factors and RNA substrates The RNA exosome and disease R-loops, RNA surveillance and genome stability  Lecture organisation Part 1: Surveillance of protein-coding messenger RNAs Introduction into mRNA degradation and key enzymes Specialised mRNA quality control pathways Nonsense-mediated decay (NMD) and genome stability Part 2: Transcriptome-wide RNA surveillance and the RNA exosome The ncRNA world The RNA exosome, its co-factors and RNA substrates The RNA exosome and disease R-loops, RNA surveillance and genome stability  Life of a eukaryotic mRNA TRANSCRIPTION DNA mRNA Protein capping, splicing polyadenylation editing RNA surveillance or quality control is important at each step: nuclear export To maintain appropriate RNA levels at any given time localisation To remove incorrectly transcribed, processed or damaged RNAs Defects in these processes and their surveillance can cause genomic TRANSLATION instability, leading to genetic diseases and cancer. STORAGE DEGRADATION  Life of a eukaryotic mRNA TRANSCRIPTION DNA mRNA Protein capping, splicing polyadenylation editing mRNAs always exist within RNA-protein complexes (mRNPs) nuclear export mRNP composition directly affects all stages: Processing localisation Localisation Translation (and its efficiency) TRANSLATION mRNA stability STORAGE DEGRADATION  Life of a eukaryotic mRNA TRANSCRIPTION capping, splicing polyadenylation editing IMPORTANT Lifetime of an mRNA is affected by how well it is translated and/or bound by the nuclear export degradation machinery. “RNA triage” localisation TRANSLATION Closed loop must be broken before exonucleases can gain access. STORAGE DEGRADATION  mRNA life-times/decay rates vary substantially Average mRNA half life in mammals ~ 8 hrs; extreme examples: c-fos 15 min, b-globin > 17 hrs RNA stability or instability sequence elements (often in 3’ UTR) t mediate interactions with the translation o Binding sites for factors that mediate interactions with the translation or degradation machineries Example: AU-rich (instability) elements (ARE) Adenine/uridine-rich elements (ARE) Confer a short half-life on mRNAs containing them – found in 5-8% of all (mammalian) mRNAs Transfer of AREs to stable RNAs (e.g. b-globin) reduces half life! AREs are bound by cellular factors, many of which interact with the RNA degradation machinery Important: there are exceptions - some ARE binding proteins stabilise transcripts (e.g. the Elav-like protein HuR) Recognition sites of general or specialised endoribonucleases mRN Died youn Example: miRNA-mediated cleavage (AGO2) in plants and mammals Ag  Eukaryotic mRNA degradation pathways Phase 1 Removal of the poly A tail (deadenylation) Decapping (Endonucleolytic cleavage to initiate breakdown) Phase 2 5’-3’ exonucleolytic digestion 3’-5’ exonucleolytic digestion https://doi.org/10.3390/biom5031515  5’-3’ mRNA degradation: XRN1 Main cytoplasmic 5’ to 3’ mRNA exonuclease Functions after decapping Involved in mRNA degradation, dependent or independent of deadenylation Member of XRN (Exoribonuclease) family of 5’-3’ exonucleases XRN1/Xrn1 (cytoplasmic): degradation of mRNAs and non-coding RNAs (ncRNAs) XRN2/Rat1 (nuclear): transcription termination, ribosomal RNA processing, nuclear degradation of (pre-) mRNAs, long ncRNAs (lncRNAs) and other ncRNAs XRN1 is found downregulated in osteosarcoma and appears to play a pivotal role during the host response to viral infection. 3’-5’ mRNA degradation: The RNA Exosome and DIS3L2 Three different exosomes in the scientific literature… https://doi.org/10.1016/j.autrev.2020.102644  3’-5’ mRNA degradation: The RNA Exosome and DIS3L2 The RNA Exosome The main 3’ to 5’ exonuclease complex in eukaryotic cells Involved in nuclear and cytoplasmic RNA turnover and processing Originally identified in yeast (RRP – ribosomal RNA processing) Multiple nuclease activities: DIS3 (nucleus) or DIS3L1 (cytoplasm) in humans (Dis3/Rrp44 in yeast) EXOSC10 (PM/Scl-100) in humans (Rrp6 in yeast) The rest of the subunits function in RNA binding and unwinding. DIS3L2 Metazoa-specific DIS3 paralogue that acts independently of the exosome in the cytoplasm Prefers uridylated substrates (involved in miRNA maturation and decay of bulk mRNA/various ncRNA) Phenotypes observed in DIS3/exosome mutants DIS3/Rrp44 “homologue of S. pombe dis3 (chromosome DISjunction)” https://doi.org/10.3390/biom5031515 Phenotypes observed in DIS3/exosome mutants DIS3/Rrp44 “homologue of S. pombe dis3 (chromosome DISjunction)” A role for DIS3 (and the exosome) in centromeric heterochromatin silencing Rapid nuclear turnover of heterochromatic transcripts reinforces transcriptional silencing. DIS3 mutations increase levels of transcripts from silent centromeric and telomeric loci. The centromere is essential for proper segregation of chromosomes, which is disrupted in DIS3 mutants. https://doi.org/10.3390/biom5031515  DIS3 paralogues and disease nuclear Myeloma-associated mutations https://doi.org/10.1007/978-3-030-19966-1 (Chapter 4); https://doi.org/10.3390/biom5031515  DIS3 paralogues and disease nuclear cytoplasmic Perlman syndrome: a rare congenital overgrowth disease that predisposes patients to cancer Mutations in genes encoding other human RNA exosome subunits cause distinct, tissue-specific diseases that can lead to cell cycle defects and apoptosis (more in second part of the lecture). https://doi.org/10.1007/978-3-030-19966-1 (Chapter 4) Nuclear RNA Processing and Surveillance https://doi.org/10.1016/j.tcb.2019.01.004 Cytoplasmic RNA Processing and Surveillance https://doi.org/10.1016/j.tcb.2019.01.004  Lecture organisation Part 1: Surveillance of protein-coding messenger RNAs Introduction into mRNA degradation and key enzymes Specialised mRNA quality control pathways Nonsense-mediated decay (NMD) and genome stability Part 2: Transcriptome-wide RNA surveillance and the RNA exosome The ncRNA world The RNA exosome, its co-factors and RNA substrates The RNA exosome and disease R-loops, RNA surveillance and genome stability  Three main mRNA quality control pathways All three pathways are directly linked to translation in the cytoplasm No-go decay (NGD) – Targets mRNAs with (structural) features that may stall ribosomes Non-stop decay (NSD) – Targets mRNAs without stop or termination codons that stall ribosomes PTC normal stop Nonsense-mediated decay (NMD) m7Gppp AAAAAAAA – Targets mRNAs with premature termination codons (PTCs) – PTCs can result from errors in transcription, splicing, editing, polyadenylation, mutations etc. – Best known as a mechanism to prevent the production of C-terminally truncated proteins that may be non-functional or even dominant negative – IMPORTANT: NMD does not only cause removal of PTC-containing, aberrant mRNAs, but also fine-tunes gene expression of “normal” mRNAs with specific features (e.g. long 3’UTR)  Three main mRNA quality control pathways All three pathways are directly linked to translation in the cytoplasm Exon-junction complex (EJC) deposited 20-24 nt 5’ of splice junctions “normal” translation termination displacement of upstream EJC NGD and NSD pathways are believed to be by the ribosome initiated during translation in the cytoplasm. EJC Many mRNAs are already marked for degradation via NMD during nuclear pre-mRNA splicing, when exon-exon junction complexes or EJCs are ribosome stalling at PTC upstream of EJC deposited onto the mRNA. no displacement of downstream EJC, NMD activation Discriminating between targets and non-targets of nonsense- mediated mRNA decay (NMD) Key NMD factors in mammalian cells UPF1 – helicase SMG1 – kinase that phosphorylates UPF1 SMG6 – endonuclease CCR4/NOT – deadenylase DCPC – decapping complex (recruited by PNCR2 and SMG5/SMG7) XRN1 and the RNA exosome TUT4/TUT7 and DIS3L2 https://doi.org/10.1038/s41580-019 Degradation of NMD targets Key NMD factors in mammalian cells UPF1 – helicase SMG1 – kinase that phosphorylates UPF1 SMG6 – endonuclease CCR4/NOT – deadenylase DCPC – decapping complex (recruited by PNCR2 and SMG5/SMG7) XRN1 and the RNA exosome TUT4/TUT7 and DIS3L2 https://doi.org/10.1038/s41580-019 Features of “normal” mRNAs that can activate NMD IMPORTANT All of these RNAs are under constant surveillance by UPF1, which is believed to bind promiscuously to all accessible RNAs. https://doi.org/10.1038/s41580-019  Lecture organisation Part 1: Surveillance of protein-coding messenger RNAs Introduction into mRNA degradation and key enzymes Specialised mRNA quality control pathways Nonsense-mediated decay (NMD) and genome stability Part 2: Transcriptome-wide RNA surveillance and the RNA exosome The ncRNA world The RNA exosome, its co-factors and RNA substrates The RNA exosome and disease R-loops, RNA surveillance and genome stability UPF1 depletion affects genome stability UPF1-depletion induces an ATR-dependent UPF1-depleted cells accumulate DNA breaks DNA-damage response stained with anti-H2AX antibody https://doi.org/10.1016/j.cub.2006.01.018 The impact of NMD factors on genome stability Indirect (via fine-tuning of gene expression): i.e. regulated cytoplasmic degradation of mRNAs that code for proteins involved in: chromosome structure and behaviour telomere maintenance DNA replication and repair chromatin-mediated silencing … Direct (via participation of NMD factors in nuclear processes): cell cycle progression DNA synthesis telomere maintenance … Many NMD factors shuttle between nucleus and cytoplasm.  DNA damage or when replication is otherwise blocked Genotoxic stress such as ionising radiation (IR) or hydroxyurea induces DNA damage and/or a block in DNA replication. Activation of various kinases act as signal transducers to phosphorylate UPF1. Ataxia-telangiectasia mutated (ATM) Ataxiatelangiectasia mutated and Rad 3-related (ATR) SMG1 DNA-dependent protein kinase (DNA-PK) Nuclear and cytoplasmic UPF1 functions ensure that histone production is closely coupled to the cellular need for newly nucleus cytoplasm synthesised chromatin. https://doi.org/10.1038/nrg2402 https://doi.org/10.1016/j.cub.2006.01.018 NMD factors are involved in RNA and DNA surveillance NMD factors are at the interface of gene and genome regulation. Non-NMD roles include: Replication-dependent histone mRNA decay at the end of S-phase or following replication stress DNA replication and repair – Interactions of UPF1 with chromatin and DNA Polymerase Telomere length homeostasis – Modulation of telomerase function – Negative regulation of the association of the telomeric ncRNA TERRA with chromatin https://doi.org/10.1038/nrg2402  Summary Part 1 – Surveillance of protein-coding messenger RNAs The major cytoplasmic mRNA degradation pathway is via deadenylation followed by 5’ end decapping and 5-3’ exonuclease (XRN1) activity. The RNA exosome or DIS3L2 degrades mRNAs from the 3’end. mRNA sequence elements and other features can affect their half lives/decay rates. Major mRNA quality control pathways (NMD, NSD, NGD) are directly linked to translation. Nonsense-mediated decay (NMD) is not only important for mRNA quality control, but it also participates in the fine-tuning of gene expression on mRNAs with specific features. Many NMD factors shuttle between nucleus and cytoplasm. NMD factors play important roles to maintain genome stability – indirectly via cytoplasmic regulation of gene expression – directly via participation in nuclear processes (DNA replication and repair, telomere length homeostasis)  Lecture organisation Part 1: Surveillance of protein-coding messenger RNAs Introduction into mRNA degradation and key enzymes Specialised mRNA quality control pathways Nonsense-mediated decay (NMD) and genome stability Part 2: Transcriptome-wide RNA surveillance and the RNA exosome The ncRNA world The RNA exosome, its co-factors and RNA substrates The RNA exosome and disease R-loops, RNA surveillance and genome stability  Eukaryotic transcriptomes require high maintenance supervision Protein-coding mRNAs Stable non-coding RNAs (ncRNAs) – Ribosomal RNAs (rRNAs), transfer RNAs (tRNAs) – Small nuclear and nucleolar RNAs (snRNAs, snoRNAs) – Signal recognition particle RNA – miRNA, piRNAs, ….. Pervasive transcription of often short-lived ncRNAs all over the genome – Yeast: CUTs (cryptic un-annotated transcripts), SUTs (stable un-annotated transcripts) – Human: PROMPTs (promoter associated transcripts), lnc/lincRNAs, and many, many others Key goals: 1. Correct RNA levels at any given time 2. Accurate co- and post-transcriptional RNA processing 3. RNA quality control at all stages  of some well-known short (and stable) ncRNAs https://doi.org/10.1038/nrg3074 From “Cellular” RNA to “Smart” RNA: Multiple roles of RNA in genome stability and beyond https://doi.org/10.1021/acs.chemrev.7b00487 Timeline of ncRNA discovery https://doi.org/10.1021/acs.chemrev.7b00487 Functions of different long ncRNA species https://doi.org/10.1038/s41580-019-0209-0  A network of eukaryotic ribonucleases 5’-3’ exonucleases endonucleases 3’-5’ exonucleases XRN1, XRN2 Exosome Exosome DICER /DROSHA DIS3L2 PIN domain proteins (SMG6) Deadenylases … The exosome complex contains 3’-5’ exonuclease AND endonuclease activities. Exosome substrates are … widespread without common structural features or sequence similarities. e.g. (pre-)mRNAs, many stable and unstable non-coding RNAs subjected to either complete degradation or accurate 3’-end processing. How does the exosome distinguish between RNAs targeted for maturation or decay?  Lecture organisation Part 1: Surveillance of protein-coding messenger RNAs Introduction into mRNA degradation and key enzymes Specialised mRNA quality control pathways QUIZ BREAK Nonsense-mediated decay (NMD) and genome stability Part 2: Transcriptome-wide RNA surveillance and the RNA exosome The ncRNA world The RNA exosome, its co-factors and RNA substrates The RNA exosome and disease R-loops, RNA surveillance and genome stability