Regulatory RNAs: A Deep Dive - PDF

Noncoding / Regulatory RNAs Mollie Meffert 421 Physiology [email protected] 410-502-2570 Learning objectives: To understand the breadth of roles played by regulatory RNAs throughout biology To know something about the discovery and analysis of these functions What do regulatory RNAs do?...

Noncoding / Regulatory RNAs Mollie Meffert 421 Physiology [email protected] 410-502-2570 Learning objectives: To understand the breadth of roles played by regulatory RNAs throughout biology To know something about the discovery and analysis of these functions What do regulatory RNAs do? RNAs regulate gene expression in all the usual ways Small (and long) RNAs in biology Bacteria have a variety of non-coding RNAs that play regulatory roles; they are typically “genes” expressed by the usual pathways in the cell including transcription and processing - Discuss tmRNA, Ribozymes, RyhB Eukaryotes have several classes (at least) of RNAs 20-30 nts in length playing diverse roles in the cell. Why was this not appreciated until the late 1990s?? - Discuss miRNAs, siRNAs, piRNAs, tsRNAs Eukaryotes also have many, many “long non-coding RNAs” truthfully about which we have very spotty information - Discuss Xist  Highly specialized function of tmRNA This ingenious RNA performs function of tRNA and mRNA and effectively rescues ribosomes stalled on “non- stop” cleaved mRNAs Tu et al. JBC (1995) 270;9322-26 (C- terminal extension of truncated recombinant proteins in Escherichia coli with a 10Sa RNA decapeptide). Keiler et al. Science (1996) 271:990-3 (Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA). Typical action by bacterial small RNA obstruction recruitment Riboswitches bind small molecules and regulate downstream events B12 riboswitch: Nahvi et al. Chem Biol (2002) 9:1043 (Genetic control by a metabolite binding mRNA). RNA as enzymes: Hammerhead Ribozyme Endo-nucleolytic self-cleavage Hydroxyl group Common themes for eukaryotic small RNAs How were they discovered (early clues)? From where do they derive? How are they processed and loaded into Argonaute family proteins? What do they do to affect gene expression? Discovery of miRNAs lin-4 lin14 and lin4 “lineage” mutants WT lin-14 (gf) phenocopied one another (Ruvkun L1 and Ambros) L2 lin14 encoded a protein whose L3 expression varied during development (high in L1 and low in L2/L3) L4 Adult terminal differentiation lin4 encoded no obvious ORF but lin14p appeared to down-regulate lin14 expression (negative regulator) lin4 encodes a small discrete RNA …expressed at the right time lin4 RNA Northern Blot …that could pair with lin14 3’ UTR Small RNAs conserved from worms to humans After this discovery the field exploded  How many miRNAs are there?? Features of miRNAs Encoded as gene (pol II usually) Fold into hairpin structure Well conserved www.miRbase.org ; MirGeneDB miRWalk2.0 TargetScan miRNAs: Humans > 2500 Drosophila 466 C. elegans 434 271 organisms represented Each miRNA has MANY potential targets (we will learn more about targets)!! **miRNAs are discrete genes Johnson KC et al, RNA 2023 Ambros, Bartel, Tuschl labs, 2001 What do precursors look like? Pri-miRNA Typically pol II transcripts (capped and polyadenylated, and can contain introns) miRNAs are sometimes found within introns - mirtrons Often found in clusters Processing involves two sequential Rnase III enzymes: Drosha in the nucleus and Dicer in the cytoplasm Ultimately yields 20+ duplex with 2 nt 3’ overhangs (what does this look like?)  RNAse III enzymes are ubiquitously involved in RNA processing “Molecular rulers” PAZ domain binds 3’ end to set length (for Dicer) Drosha interacts with DGCR8 DGCR8 (PAZ domain) Dicer has its own PAZ domain miRNA Processing steps Duplex will next be loaded into appropriate Argonaute to bring about gene regulation One strand will be retained … the “guide strand” while the other will be released … the “passenger strand” (star/asterix now often replaced by 5p and 3p) – will talk about Argonaute loading/function in a few slides  Discovery of RNAi (and siRNAs) wild-type Co-suppression Transgene-induced gene silencing: Transgene RNA Plants = Co-suppression Neurospora = Quelling Endogenous RNA Napoli, Lemieux, and Jorgensen, Plant Cell, 1990 An important observation …sense and antisense work equivalently Guo and Kemphues, Cell, 1995 The key insight … dsRNA is the silencing trigger Nature, 1998  Recapitulation of RNAi in vitro A key breakthrough: Recapitulation of RNAi in vitro using Drosophila embryos, Drosophila S2 cells, or HeLa cells Drosophila embryo lysate + dsRNA = specific degradation of target Tuschl et al., Genes and Dev, 1999 dsRNA is processed into 21-mers! Hamilton and Baulcombe, 1999 Zamore et al., 2000 Co-suppression in plants In vitro extract Sounds like a job for Dicer … Key point: most discussion of siRNAs refers to exogenously introduced RNA that feeds into the endogenous RNAi system…but dsRNA can also be generated in the cell (endogenously) Can be transient or stably expressed Dicer will process duplex in cytoplasm All versions lead to generation of “siRNAs” These are not “genes” like miRNAs But how do these RNAs function to bring about regulation of gene expression?  Mammals What are the effector molecules? Herrera-Carrillo and Berkhout, NAR 2017 Purification of RISC (in S2 cells) “RNA-induced silencing complex” Mass spec identifies Ago2 Hammond et al. 2001 Sufficient siRNA Diverse family linked to RNAi through genetics in plants, Neurospora and C. elegans Rivas et al. 2005 What do PAZ and PIWI domains do? PAZ domains bind the 3’ end of RNAs (remember Dicer) PIWI domains look like RNaseH (what does RnaseH do?) X-ray structures reveal overall Ago architecture How do duplex RNAs get “sorted” on Argonaute? RISC assembly from duplex RNAs RISC Assembly RISC Maturation Model Weaker base pairing at 5’ end of miRNA duplex favors guide selection Iwakawa et al. 2022 Schwarz et al. 2003 What do targets look like in broad strokes? miRNA siRNA Binding of miRNAs to imperfect targets leads to cleavage-independent silencing (translational repression…decay) Binding of siRNAs to perfectly complementary targets leads to cleavage at position 10-11 Notice different conformation of RNAs on Ago protein in two situations as potential explanation for distinct biochemical outcomes miRNAs use “seed” pairing to identify targets miRNAs have the ability to regulate target transcripts via cleavage or translation repression Plants – most miRNAs are perfectly complementary to targets. Animals (C. elegans, Drosophila, mammals) - virtually all miRNAs are imperfectly complementary to targets. A challenge for target prediction. lin-14 5'- UCACAACCAACUCAGGGA -3' lin-4 3'- AGUGUG AGAGUCCCU -5' A C A C Seed CU Nucleotides 2-8 are termed the “seed” sequence Prediction programs use: complementarity, stability, conservation, accessibility of site to determine strong candidates Most human transcripts likely regulated by miRNAs at some stage Often small amount of regulation by each miRNA; work in aggregate  Challenges in miRNA Target Identification Imperfect base pairing allows miRNAs to potentially interact with many targets, but also complicates target prediction Broughton et al., Mol Cell 2016 How do mammalian miRNAs silence gene expression? Gene expression is reduced (i.e. no protein made) mRNAs are decayed Argonautes bind “GW” proteins (GW182; TRNC6 paralogs), GW proteins interact with the NOT complex (involved in deadenylation), NOT1 interacts with Ddx6 (a DEAD box helicase) … not clear how they talk to the ribosome Plant miRNAs generally are perfectly complementary - siRNA-like mechanism  What drives miRNA turnover? miRNA stability varies dramatically between specific miRNAs and cellular contexts Tudor-SN can cleave and downregulate certain miRNAs (Elbarbary RA et al., Science 2017) Target-directed miRNA degradation (TDMD): now appreciated as a widespread mechanism regulating miRNA abundance. miRNAs bind to target ‘trigger’ RNAs with extended/bulged pairing to miRNA seed region, at least 6 nucleotides in the miRNA 3' end, lacking central pairing (nucleotides 9–11), results in miRNA decay instead of target repression Extended miRNA binding sites on targets that induce TDMD found in small nuclear RNAs (snRNAs), long noncoding RNAs (lncRNAs), and mRNA 3' UTRs. Other types of trigger sites able to achieve a similar outcome may be discovered in these emerging investigations. Sheu-Gruttaduaria J. et al., Mol Cell 2019 Buhagiar A.F.& Kleavland B., NAR 2024  Diverse miRNA:Target RNA Interactions: Atypical targets can turn the tables! Target-Directed miRNA degradation ~99 %