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Biologie des ARN/RNA Biology Eric Lécuyer, PhD Director, RNA Biology Lab Systems Biology Axis, IRCM Professeur-Chercheur Adjoint, Département de Biochimie Université de Montréal Associate Membre, Division of Experimental Medicine, McGill University Cours BIM-6026 16 Octobre, 2014 Presentation Outline • Overview of eukaryotic RNA populations • Biogenesis of messenger RNAs (mRNAs): capping, ~1h cleavage and polyadenylation, splicing. • Biogenesis of non-coding RNAs: ribosomal RNAs, transfer RNAs, micro RNAs, circular RNAs, long non-coding RNAs. • Functions and mechanisms of RNA Localization ~1h • Methods to Study RNA Localization • RNA Localization in Disease 2 Central Dogma of Molecular Biology Francis Crick Crick, Nature 227: 561 (1970) (1916 - 2004) Nobel Prize 1962 3 Various Categories of Eukaryotic RNAs Wahl et al. (2009) Cell, 136: 701-718. • Eukaryotic cells have diverse varieties of functional RNA molecules. • Often require specific modifications to be active: nucleolytic cleavages, sequence additions in 5’ ou 3’ of the RNA, nucleotide modifications. 4 Maturation Steps of Messenger RNAs Hieronymus et Silver (2004) Genes & Dev, 18: 2845-60. 5 Biogenesis of Messenger RNAs 6 Cap Structure at the 5’ Extremity of mRNAs Can be O2’-methylated • 7-Methyl Guanosine added via 5’-5’ triphosphate bridge. • Added before the mRNA reaches 30 nt in length by the enzyme Guanylyl Transferase. 7 Functions of the Cap Structure • Protects mARNm from degradation by 5’ 3’ exonucleases. • Contributes to the nuclear export of mRNAs. • Increases translational efficiency. 8 Cleavage and Polyadenylation the 3’ End of mRNAs • Involves 2 reactions: - Clivage 10-35nt after AAUAAA or <50nt before G/U rich sequence. - Synthesis of a poly-A chain by the Poly-A polymerase (PAP), ~200nt in length. aval 9 Functions of PolyA Tails • Protect mRNA dégradation 3’ 5’ exonucleases. by • Stimulation of tranlsation via interaction with Cap bound factors (circularization of the mARN) • Nuclear export of mRNAs. • PolyA tails are also sometimes added differnet types of noncoding RNAs (i.e. tRNA, snRNA, rRNA, snoRNA, bacterial ARN). • In prokaryotes, polyA tails can promote degradation of both coding and non-coding RNAs. 10 Eukaryotic mRNAs Undergo Extensive Splicing Prix Nobel 1993 Phillip Sharp Richard Roberts 11 Splicing is Controlled by Consensus Sequences Patel et Steitz (2003) Nat Rev Mol Cell Biol 12 Molecular Mechanism of Splicing 13 Splicing is Performed by Ribonucleoprotein Complexes that From the Spliceasome U2AF Joan Steitz Yale University 14 Transcripts Can Undergo Alternative Splicing Nilsen and Graveley (2010) Nature a) b) c) d) Use of an alternative 5’ splice site. Use of an alternative 3’ splice site. Inclusion or exclusion of ‘cassette’ exons. Exclusion ou retention of introns. 15 Cool Examples of Alternative Splicing a) b) c) Nilsen and Graveley (2010) Nature Splicing of the human KCNMA1 mRNA. The Dscam mRNA of Drosophila. The Drosophila mod(mdg4) mRNA can undergo alternative splicing in trans from exons transcribed on different DNA strands. (For each example, constitutive exons are colored in bleu et alternative exons are colored in yellow, red or violet) 16 DSCAM: an Extreme Case of Alternative Splicing DSCAM = Down Syndrome Cell Adhesion Molecule Protein involved in axon guidance. 62 Kb 7.8 Kb Domaines Ig Domaine Transmembranaire 270 KDa Mutually exclusive alternative splicing of cassette exons 4, 6, 9 et 17. Each neuron will express a different mRNA isoform. 12 x 48 x 33 x 2 = 38016 potential isoforms of DSCAM Schmucker et al (2000) Cell, 101: 671-84. 17 DSCAM Protein Prevents Interactions Between Dendrites of the Same Cell Gilbert (2010) Developmental Biology 18 Phosphorylation of the C-Terminal Domain of RBP1: An Interface to Coordinate RNA Maturation Steps Meinhart A et al. Genes Dev. 2005;19:1401-1415 The C-terminal Domain (CTD) of RPB1 of eukaryotic RNA Polymerase II: -Contains heptapeptide repeats of the YSPTSPS sequence (26x in yeast, 52x in humans) that are phosphorylated on Sérine, Thréonine et Tyrosine residues. -CTD phosphorylation is dynamic during transcription and different phosphorylation signatures allow the CTD to interact with proteins involved in RNA capping, splicing and cleavage/polyadenlyation. 19 Role of the CTD in Recruiting RNA Maturation Factors Co-Transcriptionally Phosphorylated CTD Weaver (2012) Molecular Biology 20 RNA Maturation Involves the Interplay of Trans-Acting Factors and Cis elements • Trans-regulators (green) can either be RNA Binding Proteins (RBPs) or regulatory RNAs (e.g. microRNAs). The human genome encodes >1000 RBPs (Curration from Gene Yeo’s Lab) Biogenesis of Various Non-Coding RNAs 23 Biogenesis of Ribosomal RNA (rRNAs) Non-Transcribed Sequences • Ribosomal RNA genes are clustered as repeats. • Mature rRNAs derive from an extensive-processed precursor. 24 Maturation Steps of rRNAs • The 5.8S, 18S and 28S rRNAs are produced and matured in the nucleolus, transcribed by RNA Polymerase I. • Maturation of the primary rRNA transcript requires modifications by small nucleolar RNAs (snoRNAs). SnoARNs are 60-100nt RNAs that derive from introns of mARNs. They hybridize to specific regions of pre-rRNAs and guide 2 main types of modifications: - Methylation of 2’OH of ribose. - Pseudouridylation of sprecific Uridines. • These modifications will dictate the maturation steps of the rRNA precursors. • The 5S rRNA is synthesized in the nucleoplasm by RNA polymérase III. 25 Biogenesis of Transfer RNAs (tRNAs) • Mature tRNAs are ~80nt in length and have a clover leaf secondary structure. • They are transcribed by RNA Pol III as longer precursors, sequences trimmed from 5’/3’ ends. • In eukaryotes, tRNAs are produced form individual tRNA gene loci, some will also undergo splicing. 26 Maturation of Transfer RNAs • Étape 1: coupure du côté 5’ par l’ARNase P (enzyme formé de sous-unité catalytique ARN: ribozyme). • Étape 2: clivage du côté 3’ par ARNases. (3’tARNses chez eucaryotes). • Étape 3: ajout de la séquence CCA à l’extrémité 3’ par tRNA nucléotdidyl transférase. Site ou les acides aminés seront attachés. • Modifications de plusieurs bases par méthylation. 27 Discovery of Micro RNAs (miRNAs) Victor Ambros et Rosalind Lee 28 Mode of Action of miRNAs • Partial Base Pairing: inhibits translation of the target mRNA. • Pefect Base Pairing: induces degradation of target mRNAs. (Imperfect Base Pairing) (Perfect Base Pairing) 29 IV.D. Le phénomène de l’interférence par ARN RNA Interference (RNAi) RNAi is the process by which small RNAs, either originating from exogenous or endogenous sources, can modulate gene expression, typically by causing the degradation of complementary cellular RNAs. microRNA / ‘Source Endogène’ ‘Source Exogène’ Dégradation ou répression traductionnel de l’ARN cible (vert) 30 RNAi: A Revolutionary Research Tool for Functional Genomic Screening Boutros and Ahringer (2008) Nature Review Genet, 9: 554-566 31 Long Non-Coding RNAs (lncRNAs) • LncARNs are defined as non-coding RNAs with a length >200nt. Deep sequencing studies have revealed thousands of dynamically expressed lncRNAs transcribed from the human genome. LncRNAs are often organized in close association with protein coding genes and share similar features to mRNAs (e.g. trx by RNA Pol II, capped, polyA, spliced). Mercer and Mattick (2013) NSMB, 20: 300-307. 32 Suspected Functions of LncRNAs Paralkar & Weiss (2013) Blood 33 Example of lncRNA: Xist and X Inactivation FISH sonde pour X Lee (2012) Science DAPI L’ADN du Xi est plus condensé. • In female human cells, one of the X chromosomes is inactivated (Xi) transcriptionally. This repression is modulated by the Xist RNA transcribed from the Xi chromosome This lncRNA of ~20kb coats the Xi and recruits proteins involved in heterochromatin formation. 34 Biogenesis of Circular RNAs (ciRNAs) • ciARNs and an emerging class of RNA species taht result from ‘head-to-tail’ splicing by the 5’splice junction of one exon and a donor site at the 3’end of a downstream exon. These were discovered by looking for junction reads in deep sequencing datasets. Hentze and Priess (2013) EMBO Journal, 32: 923-925 35 Possible Functions of ciRNAs Hentze and Priess (2013) EMBO Journal, 32: 923-925 36 General Principles and Functions of RNA Intracellular Localization 37 Traditional View of Protein Targeting Alternative Mechanism: Localizing the mRNA prior to translation 38 Why Localize mRNAs? • Saving energy: A single mRNA can undergo several rounds of translation. • Prevents proteins from accumulating in the wrong subcellular compartment. (e.g. MBP mRNA, oskar mRNA) • Rapid tuning of protein expression in specific subcellular compartments in response to (e.g. β-actin mRNA) • Promoting the co-translational assembly of protein complexes. (e.g. co-localization of functionally related mRNAs) 39 Biological Functions of Localized mRNAs Cody et al., 2013. WIREs Dev Biol 40 Loss of Anterior Structures in Bicoid Mutants Exosquelettes de Larves Phénotype ‘Bicaudal’ Frohnhöfer & Nüsslein-Volhard (1986) Nature • La localization antérieure de l’ARNm de bicoid est essentielle pour sa fonction et contrôlée par les séquences de son 3’UTR. 41 Anterior Nanos Targeting Causes Bicaudal Phenotype Gavis & Lehmann (1992) Cell • RNAs detected by in situ hybridization (ISH). 42 RNA Secretion Within Extracellular Vesicles Raposo et al (2013) J. Cell Biol 43 Cis-Regulatory RNA Localization Elements Van De Bor and Davis, 2004. Curr.Opin.Cell.Biol. 44 Mechanisms of RNA Localization • RNAs can be localized via distinct mechanisms, including: 1) Active transport on cytoskeletal elements; 2) Random diffusion and capture; 3) Vectorial export from the nucleus and local cytoplasmic diffusion; and 4) General degradation coupled to localized protection. (1) (3) (2) (4) Reviewed by: Lipshitz and Smibert, 2000. Curr.Opin.Cell.Biol. 45 Coding-Dependent and Coding-Independent Functions of Localized RNAs 1. Two mRNAs encoded by functionally related genes (G1 and G2). 2. Bound by RNA binding protein that recognizes common element in mRNAs. 3. mRNAs are exported from the nucleus into the cytoplasm. 4. Then they are transported to the cell cortex, where they are translated locally. Encoded proteins assemble into complex. 5. Localized mRNAs could also play coding-independent role (e.g. scaffolding function). 6. Sites of mRNA accumulation may also serve as storage depots ready Lécuyer et al., Curr Opin Cell Biol (2009) to deploy mRNA pools for local translation. 46 Mathematical Model of Localized Protein Complex Assembly Protein Half-life (min): Batada et al., 2004. PNAS • Is co-localization important for efficient assembly of protein complexes or to prevent cross-talk between pathways? • Conclusions: - Random protein interactions are rare, cross-talk is insignificant. - Co-localized sources are likely to be essential to increase the probability or protein interactions, diffusion is not enough. 47 Methods for Studying RNA Localization 48 Methods to Study mRNA Expression Localization: In Situ Hybridization (ISH) 1 Make labeled DNA or RNA probe complementary to target mRNA • Different nucleotide labels available (e.g. Dig, DNP, FITC, Biotin). • Different methods (e.g. DNA nick labeling, PCR, IVT transcription). 2 Hybridize probe to fixed and permeabilized cells, tissues or whole mount specimens. 3 Add enzyme-conjugated antibody that recognized nucleotide label. (e.g. anti-Dig antibody coupled to HRP or AP enzymes) 4 Add chromogenic or fluorescentlyconjugated enzyme substrate. (e.g. NBT-BCIP = AP substrate). 49 Fluorescent In Situ Hybridization (FISH) Posterior mRNA in Drosophila Tyramide Signal Amplification (TSA) TSA RNA CG14217 DNA NTB-BCIP DIG Lécuyer et al., Methods Mol. Biol. (2008) Lécuyer et al., CSHL Protocols (2008) 50 Diverse mRNA Localization Patterns in Fly Embryos • ~70% of mRNA are Localized during early embryogenesis, as assessed by a global FISH study of ~4000 mRNA species. 51 Lécuyer et al. (2007) Cell Correlations in mRNA/Protein Distribution DNA RNA Protein Lécuyer et al. (2007) Cell 52 Multiplex FISH for Simultaneous Detection of Multiple mRNAs Fly Patterning Genes Fly Hox Genes sog ind msh wg en lab Dfd Scr Antp Ubx abd-A abd-B Kosman et al., Science (2004) 53 Multiplex DNA-FISH for Chromosome Painting Schrock et al., 1996. Science Speicher et al., 1996. Nature Genet Method used for karyotyping human chromosomes. 54 Fluorescent In Situ Sequencing (FISSEQ) Outline: 55 Lee et al. (2014) Science, 343: 1360-1363 Assays for RNA Expression/Localization Profiling Ginart and Raj (2014) Nature Methods 56 Identifying Organelle-Enriched RNAs Separated Pseudopodia (Ps) from Cell Bodies (CB), then micro-arrays Confirmed by RT-PCR Used MS2 tagging system to show 3’UTR dependent Blower et al., J Cell Biol (2007) Mili et al., Nature (2009) Micro-array analysis reveals mRNAs associated with frog microtubules, confirmed by FISH Identified ~50 mRNAs localized to pseudopodia in fibroblasts 57 Other Global Methods to Profile Localized mRNAs Purification of RNA-binding proteins (RBPs) followed by microarrays or deep sequencing (e.g. RIP-Chip, RIP-Seq, CLIP). Keene, Nat Rev Genet (2007) Gerber et al., PLoS Biol (2004) Yeast Puf proteins associate with functionally related mRNAs 58 Methods to Image mRNAs in Live Cells Van de Bor and Davis, 2004. Curr.Opin.Cell.Biol. a) b) c) Direct injection of fluorescently labeled mRNA molecules MS2-GFP labelling system (Bertrand et al. 1998, Mol Cell) Molecular beacon technology (Bratu et al. 2003, PNAS) 59 Live Imaging of nanos mRNA in Drosophila Oocytes Via MS2 Tagging Approach MCP-GFP nanos-(MS2)6 Forrest and Gavis, 2003. Curr.Biol. Posterior targeting of nanos mRNA is established during oogenesis by 60 diffusion and entrapment mechanism New Method for RNA Live Imaging: FluorophoreBinding RNA Aptamers HBI = fluorophore 4-hydroxybenzlidene imidazoline. Paige et al (2011) Science • Used multiple rounds of in vitro selection (SELEX) to screen 5x10E13 RNA molecules (70 nt in length) to identify RNA aptamers with the capacity to bind and activate various fluorophores. 61 The Spinach Aptamer System 5S-Spinach : 5S Ribodomal RNA fused to the Spinach RNA aptamer • Addition of sucrose to the cells induces stress granule formation. Paige et al (2011) Science 62 Genetic Disorders Impacting RBPs and RNA Localization Pathways 63 Degenerative Diseases With RNP Perturbations Ramaswami M. et al. (2013) Cell 64 Synaptic Translation Often Disrupted in Neurodegenerative Disorders 65 Liu-Yesucevitz et al. (2011) J. Neurosci. Trinucleotide Repeat Expansion Diseases (TREDs) Krzyzosiak W J et al. (2011) Nucl. Acids Res. 66 Fragile X Syndrome • Most frequent inherited form of mental retardation. • Impairment ranges from learning disorders to severe cognitive disabilities. • Unusual X-linked disorder, inheritance from nonpenetrant male carriers to their non-penetrant daughters with affected sons. • Results from trinucleotide (CGG) repeat expansion within the 5’UTR of FMR1 gene. Repeats expanded ~800 times in affected individuals. • FMR1 encodes FMRP, an mRNA binding protein that regulates transport and localized translation of mRNAs. 67 FMRP Required for Stimulus-Induced mRNA Transport and Translation Dictenberg et al., Dev Cell (2008) FMRP function important for dendritic spine maturation 68 Dominant Effects of Toxic RNA Repeats in Different TREDs Krzyzosiak W J et al. (2011) Nucl. Acids Res. • Mutant CUG and CAG repeat RNAs form nuclear foci (red) in Type 1 Muscular Dystrophy (DM1) and Huntington’s disease (HD) 69 Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) • ALS and FTD share similarities in disease mechanisms, show co-morbidity. • ALS/Lou Gherig’s Disease: muscle weakness and atrophy due to motor neuron degeneration. • ALS affects 1-2/100,000 people, usually manifests from 40-60 years of age. • Both familial (10%) and sporadic (90%) cases with recurring mutations. • FTD affects 3-4/100,000 people, defined by abnormal behaviour and language. • In both ALS and FTD, neurons exhibit inclusions of aggregated proteins known as ‘Stress Granules’ that contain signature RBPs. Ling S.-C. et al. (2013) Neuron 70 Genetic Causes of ALS/FTD Robberecht and Philips (2013) Nat. Rev. Neurosci. 71 Functional Disruption of TDP43 and FUS/TLS Ling S.-C. et al. (2013) Neuron 72 What Explains Selective Sensitivity of Neurons Ramaswami M. et al. (2013) Cell 73 Myotonic Dystrophy Type I (Steinert’s Disease) •Most common form of muscle disease (1/8000 affected worldwide; 1/500 in Saguenay) •Clinical features: Multisystemic, muscle wasting and myotonia, cataracts, heart defects, catacracts, apathy, cognitive impairments. •Autosomal dominant inheritance •Expansion of CTG repeats in 3’UTR of DMPK gene on Chr 19: Normal: 5-37 repeats Pre-mutation: 38-49 repeats Symptomatic: >50 repeats 74 Expanded CUG-Repeat mRNA Sequesters Muscleblind Family Proteins in the Nucleus CUG repeats for RNA Hairpin Krzyzosiak W J et al. (2011) Nucl. Acids Res. • DMPK mRNA with long CUG repeats forms extended stem loop structure. • Aberrant RNA forms nuclear aggregates and exerts a toxic gain-of-function effect by sequestering RNA binding proteins in the nucleus. 75 Eric Wang (MIT) Chris Burge (MIT) Neal Cody Defining Transcriptome Binding Properties of Mbnl Proteins (Eric Wang) • Cross-Linking and IP and RNA sequencing (CLIP-Seq) experiments performed to define transcriptome binding profile of Mbnl proteins. Wang et al (2012) Cell, 150: 710-24. 3’UTRs Binding Properties of Mbnl1 Mbnl1 binding along 3’UTRs from CLIP datasets Enriched ‘Cellular Component’ GO categories for genes with Mbnl binding in 3’UTR Wang et al (2012) Cell, 150: 710-24. • Potential roles of Mbnl proteins in RNA localization? Wang et al (2012) Cell, 150: 710-24. (Eric Wang) Cell Fractionation and RNA Sequencing Biochemical fractionation of mouse myoblasts and fly cells, followed by RNA sequencing. (Eric Wang and Neal Cody) Wang et al (2012) Cell, 150: 710-24. Muscleblind Depletion Impacts the Targeting of Membrane mRNAs (Eric Wang and Neal Cody) Wang et al (2012) Cell, 150: 710-24. Mbnl Functions in Translation and Protein Secretion Ribosome Footprint Profiling Luciferase Assays Wang et al (2012) Cell, 150: 710-24. (Eric Wang) Mbl Functions in mRNA Splicing and Localization Wang et al., Cell (2012) Approaches to Decipher the ‘mRNA Localization Code’ Drosophila Genetics RNA Imaging in Fly and Human Cells + High-Content Screening And RNA Sequencing + Participation in ENCODE Project: Identifying Functional RNA Elements Encoded in the Human Genome K562 & Mcf7 cells Gene Yeo Xiang-Dong Fu Brent Graveley Chris Burge Eric Lecuyer Lecuyer Lab Objectives: -Defining RBP Subcellular Localization Properties - Assess impact of RBP depletion on mRNA localization 84 Fractionation-Seq to Study Transcriptome Localization Nuclear K562 Cells RNA Extraction, RiboDepletion (Ribozero) Illumina TruSeq Stranded Kit Library Generation and RNA-seq Replicates of Total, Nuclear, Cytosolic, Membrane and Insoluble fractions Wang et al (2012) Cell Western Blotting 15 Tubulin 50 KDEL 100 Ninein 190 Expression Relative to Total (%) His H3 RT-qPCR 100 75 100 100 XIST 75 ANLN 50 50 50 25 25 25 0 N C M I 0 H3 75 0 N C M I N C M I (Neal Cody & Xiaofeng Wang) 85 Broad Asymmetric Distribution of Functionally Coherent RNA Populations in K562 Cells Cell Component GO Terms M Total Transcriptome I C M LncRNA C I N P-Value (Neal Cody) 86 Interesting Examples of RNA Localization ANKRD52 (mRNA and ciRNA) Total Nuclear Cytosolic ciRNA Membrane Insoluble mRNA ANKRD52 DANCR (lncRNA) Total Nuclear Cytosolic Membrane Insoluble DANCR 87 Progress - RBP Imaging RBP Imaging Database http://csb.cs.mcgill.ca/RBPImage 88 HNRNPK / DCP1a / DAPI 89 Diverse RBP Subcellular Localization Patterns DNA Marker RBPs 90