Cavaillé Jérôme. LBME-CNRS, UMR 5099, 118 route de Narbonne Université Paul Sabatier Toulouse Cedex,

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1 Cavaillé Jérôme LBME-CNRS, UMR 5099, 118 route de Narbonne Université Paul Sabatier Toulouse Cedex, Tel : Fax : cavaille@ibcg.biotoul.fr, Web-site: grpcavaille/index.html H. Seitz, J. Cavaillé. Pour la Science (2005)

2 Concentré(e) tu resteras et 2 messages (au moins) tu retiendras 1- «Il n y a pas que les protéines dans la vie» - Codant vs Non-codant 2- «Petits mais costauds» - ARNi et mécanismes apparentés (microarn, siarn, piarn )

3 «We are more than the sum of our genes» Coding DNA < 2%) (open reading frame) Protein-coding genes (number) H.sapiens: ~ C.elegans: ~ Non-coding DNA (98%) D.melanogaster: ~ A functional role for non-coding DNA? The developmental and physiological complexity of organisms is not accompanied by a significant increase in the number of protein-coding genes Note: The diversity and complexity of the proteome is increased by alternative pre-mrna splicing and post-translational modifications.

4 The proportion of ncdna increases with «developmental complexity» 90-98% 5-20% (Mattick, 2011; see also from Taft et al., 2007)

5 The Dark Genomic matter ncdna: Cis-acting elements: promoters, enhancers, CpG islands Intergenic regions: «gene deserts» Heterochromatin domains (centromeres, telomeres «Genomic parasites»: (retro)-transposons, repeats, pseudogenes Recent high-throughput technology for transcriptome analyses (High-resolution tilling arrays, SAGE, CHiP-seq, RNA-seq, large-scale cdna cloning projects) Most, if not all, of the nucleotides of mammalian genomes are transcribed (at least in a cell- or developmental stage-specific manner) A myriad of transcripts (> ) with little or no protein-coding potential (also referred to as noncoding RNA (ncrna), or mrna-like transcripts, or non-protein coding RNA) The complexity of ncrna genes is of the same order of magnitude, if not greater, than that of the repertoire of protein-coding genes

6 Changing the paradigm: from «beeeeurk» to «Yeaaaaaaah»!

7 Back to the past! (Lac operon) Jacob & Monod (1961) J. Mol Biol 3:

8 ncrna genes - Definition ncrna genes produce transcripts that function directly as catalytic, structural or regulatory RNAs rather than encoding proteins A new hidden layer of genetic information? Experimental (or biological) noise? What about small, functional (and poorly conserved) peptides?

9 A complex repertoire of ncrna genes Small ncrnas (from ~20 to <300 nt) & Long ncrnas (from ~300 to > nt ) «Housekeeping ncrnas» (ubiquitous functions) «Regulatory ncrnas» (cell/tissue-specific functions) «Unclassified ncrnas» (poorly known functions) rrnas trnas (translation) U-snRNAs (RNA splicing) snornas (RNA modification) RNAse P/MRP (RNA processing) 7SK (Transcription). mirnas pirnas endosirnas Xist Airn Kcnqot1 ANRIL HOTAIR (RNA-mediated gene silencing) Long ncrna-mediated chromatin remodeling. -Large intergenic transcripts -Antisense RNA species -Cryptic unstable transcripts -Promoter-associated RNAs. Tiny vs Giant Poly(A+) vs Poly(A-) Spliced vs Unspliced Nuclear vs Cytoplasmic Processed vs unprocessed Ultra conserved vs Species-specific Highly vs Poorly expressed.

10 ncrnas as key players in the regulation of gene expression & cellular architecture (i) Chromosomal organization (ii) RNA processing (iii) Protein synthesis every steps in the flow of genetic information can be modulated (positively or negatively) by ncrnas

11 ncrnas function as RiboNucleoProtein (RNP) complexes ncrna + specific proteins = RiboNucleoParticle C/D snornps H/ACA snornps mirnps 7SK RNP Long ncrnas Fibrillarin NOP56 NOP kda Dyskerin GAR NOP10 NHP2 Argonaute proteins (Ago1,2,3,4) + Hexim1 P-TEFb LARP7 MePCE ~ 20% are bound by Polycomb Complex (e.g.prc2)

12 Mode of action of ncrnas (I) RNA (DNA) target «Informational molecules» (RNA guides) By base-pairing with other cellular RNA species, and also very likely with double-stranded DNA (e.g. U7-snRNA, micrornas, pirnas, endo-sirnas, snornas )

13 Mode of action of ncrnas (II) «Structural molecules» (RNA scaffold) By modulating protein activity, protein subcellular localisation or protein stability. (e.g. 7 SK, Xist and very likely many other long ncrnas )

14 Mode of action of ncrnas (III) «Functional molecules» (RNA catalysts) By catalyzing biochemical reactions (e.g. ribosomal peptidyl transferase center and peptide bond formation, self splicing introns, Ribosymes, RNAse P/tRNA maturation )

15 Mode of action of ncrnas (IV) + M «Sensor molecules» (Riboswitches) By undergoing conformational changes upon binding to specific cellular metabolites (e.g. FMN, Riboflavin )

16 De tout petits ARNs (20-30nt) extincteurs de gènes Lin4 (starn) (C.elegans) (Neurospora) Quelling Co-suppression starn & Répression de la traduction (Plantes) VIGS (C.elegans) ARN interférence Let7 (C.elegans) siarn (plante, drosophile, homme, trypanosome) > microarns (Drosophile, C.elegans, Homme, plantes) microarn/siarn miarn et Cancer? Production et 2001 mécanismes moléculaires apparentés ARNi = outil pirna et lignée germinale male rasirna, sharn & modifications de la chromatine

17 «le papier fondateur» en «la découverte de l année» en le prix Nobel en Andrew Z. Fire (left), and Craig C. Mello (right)

18 : L addition d une copie supplémentaire d un gène codant pour une pigmentation violette (chez le pétunia) s accompagne non pas de l apparition de pétunias plus violets mais au contraire de pétunias.blancs: Mise en silence de la copie exogène mais aussi de la copie endogène!!!

19 Injection d un ARN db homologue au gène A ( pb) Perte de la fonction du gène A C.elegans ARN interférence (ARNi): -Effet spécifique de l homologie de séquence (appariement de bases) -Importance de la structure double brin (Brin sens ou antisens :peu/pas d effet) - Efficace à doses sub-stoechiométriques (ARNdb < ARNm du gène cible) -Effet systématique de tissus à tissus (amplification et/ou catalyse) - Evénement cytoplasmique post-transcriptionnel (Peu/pas d effet si homologie avec un intron)

20 Co-suppression/PTGS antisens sens Prix Lasker lignées de tomates transgéniques: Insertion de l ADNc ACO (1-aminocyclopropane-1-carboxylate oxidase sous le contrôle sous le contrôle du promoteur viral 35S (Cauliflower)

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22 Small interfering ARN ( siarn ) & ARN interférence (ARNi) Précurseur: ARNdb Dicer: endonucléase siarns Maturation à partir d'un précurseur ARN db siarn PAZ (Equipe Hannon, 2002) Phase d'initiation Double brin, 21 nt (extrémités 3 sortantes (2 nt) (Hamilton, 1999) Argonautes RISC (minimal 160 kda, holo-risc 80S) _Argonautes _Fragile X mental retardation protein 1 _Vasa intronic gene _Tudor nuclease PAZ Clivage séquence spécifique: ARNi Piwi (Equipe Tuschl, 2002) (Equipe Hannon, 2002) Phase effectrice ARNi Dégradation d'arnm homologue Processus conservé Cytoplasmique ARNm cible (Ago-2 clive au centre de l hybride miarn:arnm (Ago-2 clive le brin passager, non incorporé)

23 1: Ago are expressed ubiquitously 2: Piwi are restricted in germ cells and stem cells

24 Caractéristiques du siarn: - 2 nucléotides non-appariés aux extrémités 3 - Extrémité 5 monophosphate et 3 OH - Le brin dont l extrémité 5 est la «moins» appariée (d un point de vue thermodynamique) incorpore RISC Chez les plantes, C. elegans et S. pombe Amplification du signal (siarn= primer pour une activité ARN polymérase Arn dépendante, RdRP)

25 Jaune : perte partielle GFP Rouge: perte totale GFP - Passage de cellules à cellules (tissus à tissus) - Amplification du signal ARN initial (ARN polymérase ARN dépendante)

26 Interférence ARN (ARNi) et protection contre les parasites génomiques (rétro-transposon, virus.) 1) Arguments génétiques: Mobilisation accrue des transposons dans les souches de C. elegans mutées dans des gènes impliqués dans la voie de l ARNi 2) Arguments moléculaires: Clonage de siarn homologues à des séquences de (retro)transposons, virus 3) Arguments évolutifs : Les virus de plantes possèdent des gènes dont le produit inhibe l ARNi (suppresseurs de mise en silence). «Guerre moléculaire» entre parasites et cellules hotes Co-évolution Ex: le suppresseur viral P19 séquestre le siarn

27 siarn synthétique contre lamin A/AC

28 L interférence par l ARN: un outil pour invalider l expression des gènes Spécificité? Réponse Interféron? Toxicité? Effet(s) secondaires? Vectorisation?

29 De tout petits ARNs (20-30nt) extincteurs de gènes starn & Répression de la traduction Lin4 (starn) (C.elegans) 1993 (Ambros, 1993) (Neurospora) Quelling (Plantes) VIGS (C.elegans) ARN interférence (Fire,1998) Let7 (C.elegans) > microarns (Drosophile, C.elegans, Homme, plantes) 2000 microarn/siarn (Ruvkun, 1993) Production et 2001 mécanismes moléculaires apparentés (Hannon,2001) siarn (plante, drosophile, homme, trypanosome) (Baulcombe, 1999) ARNi = outil (Tuschl) miarn et Cancer? mirna et maladie(s)? pirna et lignée germinale male rasirna, sharn & modifications de la chromatine (Martienssen, Grewal, Allshire, 2002)

30 Ou l on apprend que la génétique identifie un microarn dans «l indifférence générale» ou presque Prix Lasker 2008

31 Heterochronie et développement larvaire de C. elegans

32 La paradigme Une régulation post-transcriptionnelle Le locus lin-4 génère L1 L2 L1 L2 L1 L2 L1 L2 Expression de lin-4 L1: - L2: + Protéine ARNm deux petits ARN (~ 20 et 65 nt) médiée par la région 3 UTR de lin-14 (appariement avec lin-4)

33 Let-7

34 Lin-4 and Let-7 = Small temporal RNAs = micrornas (21-23 nt) starn = microarn

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36 Un microcosme à ARN ~250 ~850 ~ Aujourd hui: 678 microarn humains (la limite supérieure?) (e.g. mirnas retrouvés uniquement chez les primates) (version 11.0, April 2008)

37 De tout petits ARNs (20-30nt) extincteurs de gènes starn & Répression de la traduction Lin4 (starn) (C.elegans) 1993 (Ambros, 1993) (Neurospora) Quelling (Plantes) VIGS (C.elegans) ARN interférence (Fire,1998) Let7 (C.elegans) > microarns (Drosophile, C.elegans, Homme, plantes) 2000 microarn/siarn (Ruvkun, 1993) Production et 2001 mécanismes moléculaires apparentés (Hannon,2001) siarn (plante, drosophile, homme, trypanosome) (Baulcombe, 1999) ARNi = outil (Tuschl) miarn et Cancer? mirna et maladie(s)? pirna et lignée germinale male rasirna, sharn & modifications de la chromatine (Martienssen, Grewal, Allshire, 2002)

38 Drosha Dicer Dicer: pre-mirna mirna Drosha: pri-mirna pre-mirna Ketting et al., Genes and Dev 2001 (Cf. aussi Hutvagner et al., Science 2001 et Grishok et al., Cell 2001) Lee et al., Nature 2003 (Cf. aussi Denli et al. Nature 2004)

39 Du pri-miarn vers le pré-miarn

40 . du pré-mirna vers le mirna.

41 Animaux Plantes

42 Organisation génomique des microarn Berezikov (2011) Nature Reviews Genetics

43 Yang et al (2011) Mol Cell

44 Production de miarn-like via des petits ARNnc Yang et al (2011), Mol Cell

45 Régulation de la maturation des pri-(pre-)mirnas Winter et al (2009) Nature Cell Biol

46 Régulation de la maturation des pri-(pre-)mirnas Winter et al (2009) Nature Cell Biol

47 Argonaute proteins are classified into 3 paralogous groups Affinity for single stranded 3 -ends and duplex sirna-like ends 5 -binding pocket (5 P nucleotide of the guide RNA strand) C.elegans-specific group Slicer catalysis site (RNAse H-like fold) Argonaute-like proteins (AGO) Piwi-like proteins (PIWI) Ago are expressed ubiquitously/piwi are restricted in germ cells and stem cells Hutvagner and Simard, 2007, Nature Reviews Molecular Cell Biology; Parker and Barford, 2006, TIBS

48 MicroARNS & inhibition de la traduction (ou ARNi) Drosha-DGCR8 (nucleoplasme) Produit primaire de la transcription ~ kb-long = pri-mirna ARN précurseur de 70 nt en tige boucle = pre-miarn Dicer-TRBP (cytoplasme) microarn Phase d'initiation Argonautes and Co micrornp ARN interférence!! ARNm cible Inhibition de la traduction Phase effectrice (complémentarité parfaite) (complémentarité partielle) Ago-2 Ago1-4 (Plantes) (Animaux) Pas si simple!!! Cf Brodersen et al., Science 2008

49 «Comment ça marche»? Filipowicz et al.,nat Rev Genet (2008); Eulalio et al., cell (2008); Wu and Belasco; Molecular cell (2008); Richter et al., Nature Struc & Mol Biol (2008).

50 miarn et P-bodies (P-bodies) mrnp-mirnps s accumulent dans P-bodies Cause? Conséquence?

51 Computational identification of mirnaregulated genes Web-sites

52 Micromanagers of gene expression #- Vertebrate genomes may encode ~ 300-(1000?) different mirnas # % of mrna = targets? (in silico predictions)

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56 Les miarn «façonnent» la structure du transcriptome Kloosterman et Plasterk, Developmental Cell, 2006 Bartel et Cheng, Nature Reviews Genetics, 2004

57 Hypothesis: mirna repress leaky transcripts and buffer genetic noise (confer robustness to developmental genetic programs) Ex:«They maintain the fidelity of developemental programs rather than initiating developemnatl transitions»

58 Des miarn et des boucles

59 Transcription versus Post-transcription Evolution (mirnas ~ facteurs de transcription) Rapidité d exécution Caractère réversible (mirnas > facteurs de transcription)

60 Des miarn chez l anemone (N vectensis) Grimson et al. Nature 2008

61 microarn et Evolution 1- De nouvelles structures anatomiques 2- De nouveaux réseaux de régulation génique 3- Variabilité phénotypique Berezikov (2011) Nature Reviews Genetics

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63 Fonctions biologiques des miarn (part I)

64 Fonctions biologiques des miarn (part II) Park et al (2010) Hum Mol Genet

65 miarn et stades larvaires (C. elegans)

66 miarn et Apoptose

67 Neurones Muscles Sang Coeur

68 Cellules ES

69 microarn et développement précoce

70 mirnas et virus

71 mirnas and Cancers (I) Many human mirna genes (> 50 %) are located at sites known to be involved in cancers (fragile sites, minimal region of loss of heterozygoty, breakpoint regions..) Ex: mir polycistron is located on Chr.13q32-33 in a region that is amplified in follicular lymphoma. micrornas as Oncogenes? mir-15 and mir-16 are located within a 30 kb deletion in chronic lymphocytic leukemia microrna as tumour suppressors? Calin et al PNAS, Croce et al 2009, Nature Reviews Genetics

72 mirnas and Cancers (II) Many mirnas are differentially expressed in different cancers mir-15, mir-16: Down-regulated in B-cell chronic lymphocyte leukemia mir-143, mir-145: Dow-nregulated in colorectal neoplasia mir-155/bic: Up-regulated in diffuse large B-cell lymphoma (X 100!!) mir-17-mir-92 cluster: Up-regulated in B-cell lymphoma let-7: Down-regulated in lung cell carcinoma mir-125b: Down-regulated in breast cancer mir-221: Up-regulated in glioblastoma mir-128, mir-181a,b,c: Down-regulated in glioblastoma Van den Berg et al., (2003) Genes, Chromosome & Cancer; Calin et al., (2002) PNAS 99: PNAS (2004) 101: ; Eis et al., (2005) PNAS ; Iorio et al., (2005) Cancer Res Michael et al., (2003) Molecular cancer Research 1: ; O Donnel et al., (2005) Nature 435: A causal role in the generation or maintenance of tumours?

73 mirnas and Cancers (III) Lu et al., (2005) Nature 435: mirna profiles reflect the developmental lineage and differentiation state of Tumors «unlike with mrna expression, a modest number of mirnas (~200 in total) might be sufficient to classify human cancers.»

74 mirnas and Cancers (IV) He et al., (2005) Nature 435: Overexpression of the mir-17-19b (OncomiR) cluster accelerates c-myc-induced lymphomagenesis in mice ---Decrease in the latency of leukaemia development (from 3-6 months to 51 days) ---Increase in the frequency of the cancer (30 % to 100 %)

75 microrna et cancers: Oncogenes ou suppresseurs de tumeurs (V) Croce et al 2009, Nature Reviews Genetics

76 Et c est reparti! (pirnas)

77 pirnas (Piwi-interacting RNAs) - pirnas: ~ nt long - with a 5 monop group, (preference for U) and 2-0-methylation at their 3 end - In contrast to mirnas, pirnas are poorly conserved (even between closely related species) -Associated with PIWI proteins «The ping pong model» - Biosynthesis? -They are processed from single strand precursor in a process independent of Drosha and Dicer. - Expression? -They are abundant in teste/germ cells: 1_piRNAs at pachytene stages (> RNA species)- clustered in loci containing small RNAs) 2_piRNAs before the pachytene stage match repeated sequences (re)transposons -Function(s)? Transposon silencing (de novo methylation) Germ cell development Aravin et al., (2007) Science, Stefani et al., (2008) Nature Reviews Mol and Cell Biol

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79 A model of pirna biogenesis

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81 Juliano et al (2011) Annual Review of Genetics

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