RNA Processing 2024 PDF

RNA Processing Molecular Biology and Genomics Course Luisa Cochella, PhD Multiple RNA species are at the heart of the central dogma snoRNA rRN snRNA A DNA mRNA tRN Protein A miRNA...

RNA Processing Molecular Biology and Genomics Course Luisa Cochella, PhD Multiple RNA species are at the heart of the central dogma snoRNA rRN snRNA A DNA mRNA tRN Protein A miRNA piRNA siRNA The central dogma within the cell Nucleus: DNA replication Transcription DNA replication RNA Transcription processing RNA RNA decay processing Translation RNA decay Cytoplasm: RNA processing Translation Different RNA polymerases make distinct products Eukaryotes: RNA pol I rRNA RNA pol II mRNAs, snRNAs, snoRNAs, miRNAs RNA pol III tRNAs, 5S rRNA, U6, other small RNAs The product of transcription is only the beginning Primary Mature RNA transcript Cleavage Splicing Terminal nucleotide addition Editing (insertion, deletion, modification) Most RNAs require multiple processing steps Precursor Processing steps Mature Polycistroni Multiple cleavage events 5S, 16S, 23S c rRNA Nucleotide modifications (Bact.) 5.8S, 18S, 25S (Euk.) Precursor Cleavage, splicing, terminal tRNA tRNA nucleotide addition (CCA), base modifications Pre-mRNA Capping, splicing, base mRNA modifications, polyadenylation Small RNA Endonuclease cleavage, exonuclease miRNAs, piRNAs, Ribosomal RNA transcription, processing and assembly are coupled Ribosomal RNA (rRNA) transcription Miller and Beatty Science, 1969 10-44 kb regions, repeated in all genomes “polycistronic” RNAs: enables equimolar production of various rRNAs pol I transcripts (very strong promoter) 1% of genome but 40% of transcription rRNA processing, a multi-step pathway Co-transcriptional association with processing factors and ribosomal proteins (RPs) Multiple endonucleases (and exonucleases in higher eukaryotes) release rRNAs Dynamic association and dissociation of processing factors as rRNAs fold and bind RPs Mostly in the nucleolus, some maturation continues in the nucleoplasm RNase III domains are key for processing rRNA and other RNAs Dimer of a bacterial RNase III dsRB D RIIID Mg2+ dependent endonucleases Cleave dsRNA and intramolecular stems (e.g. in pre rRNA, snoRNAs and snRNAs) dsRBD Leave 2 nt, 3′ overhangs RIIID Generate ends with 5′ PO4 and 3′ OH Court DL et al. Annu Rev Genet 2013 Transfer RNA (tRNA) processing CCA adding RNaseP RNaseZ enzyme removes 5′ removes adds new 3′ end leader 3′ trailer Various nucleotide modifying enzymes TSEN complex excises introns RNase P is a RNP and one of the first discovered Bacteria ribozymes Archea * Eukarya Wu J et al. Cell 2018 * by Sydney Altman, Norman Pace and RNA processing enzymes enable a wealth of applications Xie K. et al. PNAS 2015 RNA processing enzymes enable a wealth of applications Xie K. et al. PNAS 2015 Key Points Precise ends of mature mRNAs and tRNAs (and most RNAs) are generated by cleavage by RNases Ends are primarily defined by endonucleases, but exonucleases can be used to refine them Many RNases rely on structure for specificity 3′ terminal nucleotide addition and removal of intervening sequences is important for tRNA maturation RNases (and other RNA processing enzymes) can be repurposed for diverse applications rRNAs and tRNAs have many modified nucleotides Human 28S rRNA Human tRNA Ribonucleotides can be modified in >100 manners Base modifications Ribose modifications Nucleotide modifications have diverse consequences Modulation of base-pairing & Structure Binding of specific decoding stabilization “readers” 2′OMe affects structure and stability Guiding modification specificity on rRNA Methyltransferase PseudoU synthase Nop1p/fibrillarin Cbf5p/dyskerin snoRNPs (snoRNAs + proteins) tRNA modifying enzymes use structure and sequence for specificity Pseudo Uridine Synthases (PUS) Methyltransferases (TRMT, NSUN, METTL) Adenosine Deaminases (ADAT) Chujo & Tomizawa. FEBS Key Points Large fractions of nucleotides in rRNA and tRNA are post- transcriptionally modified (up to 25% in tRNA!) Methylation of the 2′ OH and pseudouridylation are the most common, but there’s a large diversity Specificity of rRNA modifications is determined by base pairing interactions with snoRNAs that guide respective enzymes Specificity of tRNA modifications relies primarily on global and local structure. These modifications can be very elaborate and require a series of steps Nucleotide modifications affect structure, stability and the ability to interact with other RNAs and proteins Most RNAs require multiple processing steps Precursor Processing steps Mature Polycistroni Multiple cleavage events 5S, 16S, 23S c rRNA Nucleotide modifications (Bact.) 5.8S, 18S, 25S (Euk.) Precursor Cleavage, splicing, terminal tRNA tRNA nucleotide addition (CCA), base modifications Pre-mRNA Capping, splicing, base mRNA modifications, polyadenylation Small RNA Endonuclease cleavage, exonuclease miRNAs, piRNAs, Eukaryotic messenger RNA (mRNA) processing Pre-mRNA Mature 5′ capping mRNA Splicing 3′ cleavage & polyadenylation Base modifications Pre-mRNA capping mRNA 3′ end formation: cleavage & polyadenylation CPSF CstF PABP A convenient handle for mRNA enrichment *Only ~3% of total cellular RNA is mRNA Histone mRNA 3′ end formation - an important Histone exception mRNAs lack polyA tail Are short lived and regulated by cell cycle 3′ end stem loop is generated by U7 snRNP-mediated reaction Stem loop bound by SLBP Roles of the 5′ cap and polyA tail Help direct downstream maturation including splicing and transcription termination Important for transport to cytoplasm Stabilize mRNA Important for translation initiation Gallie DR. Genes & Dev, 1991 Key Points mRNA ends are defined by capping and cleavage & polyadenylation These are necessary for stability but also for downstream events Defining features of mRNAs are helpful for biochemical distinction from other RNAs for downstream analysis Pre-mRNA splicing Chicken alpha 2 (type I) collagen gene 37 Kb, >50 introns Ohkubo H et al. PNAS 1980 *Richard Roberts and Phil Sharp won the Nobel Prize for this Intron content differs substantially across species ~228 total introns in yeast % of genes with X introns human Titin gene has 363 introns! human Dystrophin gene has Number of introns 79 introns (98% of its 2.4 mb) Intron lengths differ substantially across species yeast genes have infrequent short introns human genes have short exons and frequent long introns What makes an intron? The splicing reaction sequential trans-esterification reactions products are ligated exons and a branched lariat (2’5’) intron  The spliceosome catalyzes pre-mRNA splicing The spliceosome is composed of five snRNPs And two other protein complexes Plaschka et al. Cold Spring Harb Perspect Biol, 2019 Spliceosome assembly begins with U1 and U2 binding U1 U 2 The spliceosome is dynamically assembled over every intron U1 U 2 Multiple helicases (using energy from ATP) enable large rearrangements of RNA:RNA interactions The spliceosome is dynamically assembled over every intron Plaschka, Lin & Nagai. Nature, 2017 Consensus sequences are necessary but not sufficient… Exonic splicing enhancers (ESEs) are recognized by SR proteins (Ser/Arg rich proteins with RRM domains) Intronic Splicing Enhancers (ISEs) Exonic and Intronic Splicing Silencers (ESSs and ISSs) have regulatory roles and help avoid use of cryptic sites – often bound by hnRNPs (heterogeneous ribonucleoprotein particles) An important consequence of splicing: EJC deposition EJC = Exon Junction Complex a “mark” of correct splicing The spliceosome is yet another remnant of the RNA world group I introns group II introns pre-mRNA introns Bacteria Bacteria Eukaryotes Archea Archea Fungal/plant Fungal/plant mitochondria mitochondria and chloroplasts and chloroplasts Self-splicing Spliceosome-mediated Self-splicing Requires ATP (helicases) Requires GTP Intron-lariat intermediate Intron-lariat intermediate Linear intermediate Common ancestor Key Points Introns are common in eukaryotic genes and must be removed from pre-mRNA to produce mRNA Introns are marked by a 5’ splice site, branch point sequence, and 3’ splice site Splicing proceeds in a two-step transesterification reaction The spliceosome catalyzes intron removal Splicing is a multi-step process involving more than 80 proteins, and 5 snRNAs RNA helicases and step-specific splicing factors assist with remodeling of the protein and RNA network during the different steps The spliceosome is a protein directed ribozyme Alternative processing is frequent and plays regulatory roles Alternative splicing – skipped exon Alternative 5’ splice site Alternative 3’ splice site Alternative transcriptional start site Alternative polyadenylation site  Extreme cases of alternative processing rosophila Dscam (Down syndrome cell adhesion molecule) 38,016 splice isoforms Mammalian Pcdh (Clustered protocadherins) Multiple alternative initiation isoforms Jin & Li. Cell Mol Life Sci, 2018 Splicing control – a game changing therapy Spinal Muscular Atrophy (SMA) is caused by low SMN levels *SMN is a chaperone that facilitates assembly of snRNPs Work from Adrian Krainer, Ravindra Singh and Elliot Androphy’s la Splicing control – a game changing therapy Modified backbones for stability, specificity and delivery First FDA approved drug for SMA treatment Hua et al. (Krainer lab). AJHG, 2008 Key Points Alternative processing (splicing, initiation or cleavage and polyadenylation) are common and result in multiple isoforms per gene These processes have been particularly exploited to enable complexity of the nervous system Splice site selection requires positive and negative regulators, in addition to the necessary consensus sequences. These additional elements can be regulated to result in alternative spliced mRNAs The speed of transcription also affects splice site selection It is possible to manipulate splicing with antisense oligos for therapy. But also with small molecules! (current therapy for SMA is the small molecule Risdiplam) Eukaryotic mRNAs also carry modified nucleotides tRNA modifying enzymes ADAR1: mostly editing of endogenous dsRNA prevents immune activation ADAR2: Specific editing events recode important mRNAs (e.g. mRNA modifications enable self vs. non-self recognition Production of a pro-inflammatory cytokine Markers of immune cell activation Karikó, Buckstein, Ni & Weissman. Immunity, 2005 Key Points Base modifications are also common in mRNA Editing by ADARs is best understood m6A is abundant and many functions have been proposed Other modifications are less abundant and may represent “spill over” activity from tRNA modifying enzymes Multiple modifications seem to mark mRNAs as “self” to prevent immune activation Most RNAs require multiple processing steps Precursor Processing steps Mature Polycistroni Multiple cleavage events 5S, 16S, 23S c rRNA Nucleotide modifications (Bact.) 5.8S, 18S, 25S (Euk.) Precursor Cleavage, splicing, terminal tRNA tRNA nucleotide addition (CCA), base modifications Pre-mRNA Capping, splicing, base mRNA modifications, polyadenylation Small RNA Endonuclease cleavage, exonuclease miRNAs, piRNAs, Completing the RNA life cycle – RNA decay Transcriptio Translation n DNA mRNA Protein Decay Decay RNA decay has different purposes and uses distinct mechanisms RNA turnover to maintain steady-state or for regulatory purposes Degradation of defective RNAs (quality control pathways) Removal of foreign RNA (CRISPR, RNAi, protein-based foreign RNA recognition) Average half-lives of different RNA molecules Bacterial mRNA decay Belasco. Nat Rev Mol Cell Biol, 2010 Eukaryotic mRNA decay *Rates of deadenylation can be regulated by mRNA structure or sequence mostly at the 3’UTR (protein or miRNA binding sites) Passmore & Coller. Nat Rev MCB, 2021 Key Points RNA decay is critical to steady-state RNA levels (and is tightly regulated) Bacterial mRNA decay needs endonucleolytic cleavage Eukaryotic mRNA decay begins with deadenylation, followed by decapping and finally exonucleolytic cleavage from the ends (XRN1 and exosome) The rate of deadenylation and decapping can be regulated by 3’ UTR structure and sequence (through binding to other factors) Consequences of RNA processing Production of functional RNAs and RNPs But also… Opportunity for regulation (concentration, rate) Generation of molecular diversity Biogenesis quality control Why is eukaryotic gene expression so complex? Evolution of organismal complexity is a tempting answer, but: All of these mechanisms appeared in unicellular eukaryotes… Massive abundance of prokaryotes says these “embellishments” are not necessary for the central dogma per se A hypothesis for the evolution of this complexity Chromatin obstructs transcription (and restricts access to DNA) A nuclear envelope acts as a gatekeeper of the genome Splicing, capping, polyadenylation are necessary for nuclear export and translation (i.d. cards for endogenous mRNAs) RNA modifications mark endogenous RNA They likely evolved as part of an arms race against genomic Madhani H. The frustrated gene. Cell, 2013 parasites!