Régulation génétique par les petits ARN non-codants

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1 Régulation génétique par les petits ARN non-codants
Institut Pasteur « Génétique de la souris » 27 janvier (1h30) Régulation génétique par les petits ARN non-codants Christophe ANTONIEWSKI

2 Découverte de l’interférence par l’ARN

3 Post Trancriptional Gene Silencing
Interférence par l’ARN feeding transfection Injection Viruses ARN double-brin Post Trancriptional Gene Silencing (dégradation de l’ARNm de séquence homologue) Exogenous Endogenous Inverted-repeat transcription Homology dependent Cosuppression

4 1ère partie: siRNA (small interfering RNA)
Luc dsRNA* Cyc dsRNA* S2 Extract Dicer Immunoprecipitation Bernstein et al. Nature (2001) 409: 363 Elbashir et al. Genes & Development (2001) 15:

5 Caractéristiques des siRNA
(O-Met-2’) (2’-O-Met) 19 nucléotides double-brin 2 nucléotides débordants en 3’(OH) Activité RNAi per se Activité séquence-spécifique Ciblent ARN sens et antisens 2’-O-Met (in vivo)

6 Le complexe RISC (RNA Induced Silencing Complex)
Williams RW, Rubin GM PNAS 2002 Argonautes Deux groupes de protéines selon la présence d’aa conservés N-term: AGO-1, AGO-2 : ubiquitaires Piwi, Aubergine, AGO-3: Lignée Germinale

7

8 RNA Induced Silencing Complex RISC
mRNA degradation Helicase ? (Armitage/sde3) DG DG R2D2 se fixe à l'extrémité la + stable Recrutant Dcr à l'extrémité la - stable R2D2 "reconnaît" le 5'P du brin "passager" Cleavage and release of the "passenger" strand

9 2ème partie: les microRNA
Lin-14, lin28 Lin-41, hbl1 lin-4 lin14 CDS 3’ UTR

10 Ordres de grandeur Genome miRNA identifiés expérimentalement C. elegans D. melanogaster M. musculus H. sapiens ~150 ~700

11 Structure des gènes des miRNAs - primary miRNA (pri-miRNA)
« Cappés » Queue Poly-A Pol II-dépendants miRNA autonomes ~50% ~50% miRNA "passagers" Rare

12 Pri-miRNA --> Pré-miRNA
Seitz and Zamore Cell 125, p827, June 2, 2006 Han et al. Cell 125, 887–901, June 2, 2006

13 Pri-miRNA --> Pré-miRNA : le complexe « microprocessor »
Seitz and Zamore Cell 125, p827, June 2, 2006 Han et al. Cell 125, 887–901, June 2, 2006

14 Biogenèse des miRNA 1 G Biais 5’U RISC Mais quelle Argonaute ?

15 « specialized » miRNA and siRNA pathways in Drosophila

16 Répression traductionnelle Vs clivage
Saxena et al JBC 2003

17 La séquence « graine » (seed)
Doench et al G&D 2004 Stark et al Plos Biol 2004  Dur de trouver une cible de miRNA !

18 Les miRNA agissent aussi en déstabilisant les mRNA cibles
Lim et al. (2005) NATURE VOL 433, p770 miR124 fortement exprimé dans le cerveau  Transfection de miR 124 dans des cellules Hela  174 gènes réprimés (Niveau ARN)

19 Modèles d’action des miRNA
Eulalio et al, Cell 2008

20 Fonction des miRNAs au cours du développement
Adapted from Alvarez-Garcia and Miska, Development 2005

21 miRNA et Cancers miR17 Amplified in B Cell lymphoma
cMyc target + regulates E2F1 Trangenic mice overexpression miR17 harbor c-myc-induced lymphomas They target Bcl2, upregulated in many types of cancers Classification of human cancers using miRNA profiling Lu et al (2005). Nature 435, 834

22 3ème partie: les piRNA 25 paires de bases
Biaisés en 5’: U (80% des cas) 2’-O-méthylés à l'extrémité 3’ Correspondent majoritairement à des éléments transposables (transposons et rétrotransposons)  rasiRNA surtout abondants dans la lignée germinale (ovaires et testicules) Bergman et al. Genome Biology 2006, 7:R112 Aravin et al. Developmental Cell (2003), Vol. 5, 337–350.

23 piRNAs Co-immunoprécipitation par anti-Piwi
À partir d’extraits d’ovaires Clonage/séquençage des petits RNA associés Les piRNAs représentent 80% des petits ARN coprécipités avec un Anti-Piwi Il correspondent bien à des éléments répétés du génome (rasiRNA). Saito et al., Genes and Dev (2006)

24 Les piRNA sont majoritairement « antisens »
Saito et al., Genes and Dev (2006)

25 Les acteurs importants pour le silencing des rétroéléments sont
Dans la lignée germinale, les piRNA répriment l’expression des Elements Transposables Vagin et al., Science (2006) La voie classique RNA-i est sans effet sur le contrôle des rétroéléments Pas de contrôle par dcr1 sur mdg1, roo et mst40: la voie miRNA est également sans effet Les acteurs importants pour le silencing des rétroéléments sont Piwi, Aubergine, armi, et Spn-E (Homeless)

26 Séquençage direct de molécules uniques
Pyroséquençage « 4-5-4 » From Margulies, M., et al. (2005). Nature 437, Solexa, Solid, Helico… 10 à 100 millions de lectures, de ~ 50 nt

27 Pyroséquençage… Et la lumière fut !

28 Origine génomique des loci producteurs de piRNA

29 Biogenèse et mécanisme d’action des piRNA chez la drosophile

30 Deep sequencing of Ago2-associated small RNAs
Now, I need to briefly remind you that there is currently 3 distinct small RNA pathways characterized in this model organism. In the microRNA pathway, small stem loop RNA molecule are cropped from primary long RNA by the Rnase Drosha and exported in the cytoplasm by an exportin 5 dependent system. These pre-miRNA are then processed by Dicer-1 and one strand of the miRNA duplex is then loaded into an Argonaute-1 complex which mediates inhibition of translation and destabilization of the target mRNA. Long dsRNA, as I just shown you for viral genomes, are processed by Dicer-2, and loaded as single stranded siRNA into the argonaute-2 RISC complex which mediates an endonucleolytic cleavage of the target mRNA. It has been recently demonstrated by the Hannon and Siomi teams that a high level of small RNA is produced in the drosophila germ line from transposon repeats in the pericentromeric regions of the chromosomes. We now know that transposon transcripts are processed by a complex Ping-Pong mechanism, which is Dicer independent, and involves the three germ line specific Argonaute proteins, Piwi, Aubergin and Argonaute-3. I will not go in detail in this pathway. Just remind that in Drosophla, the reapeat associated rasiRNA are also piRNAs because they are found associated to these three Argonautes of the PIWI clade. A last word will be to say that piRNAs are though to mediate transponson silencing at the post transcriptional level, through the endonucleolytic activity of the piwi, aub and ago3 proteins. There is a forth small RNA pathway which as been pretty well characterized in pombe and plants by remains elusive in drosophila. Though this pathway, small RNAs arising from transposon repeats direct the methylation of the H3-H9 histone residue and thereby participate to the recruitment of the heterochromatin protein 1 and heterochromatin formation. The proviral activity of the suppressors in flies prompted us to study whether they can perturb one or the other of these known pathways and in particular whether they can perturb chromatin dynamics.

31 Biogenèse des endo-siRNA: 3 type de loci producteurs
Les éléments répétés, transposons et rétrotransposons Des ARN non-codants « structurés » Les zones de recouvrement des 3’ UTR (gènes convergents) Now, I need to briefly remind you that there is currently 3 distinct small RNA pathways characterized in this model organism. In the microRNA pathway, small stem loop RNA molecule are cropped from primary long RNA by the Rnase Drosha and exported in the cytoplasm by an exportin 5 dependent system. These pre-miRNA are then processed by Dicer-1 and one strand of the miRNA duplex is then loaded into an Argonaute-1 complex which mediates inhibition of translation and destabilization of the target mRNA. Long dsRNA, as I just shown you for viral genomes, are processed by Dicer-2, and loaded as single stranded siRNA into the argonaute-2 RISC complex which mediates an endonucleolytic cleavage of the target mRNA. It has been recently demonstrated by the Hannon and Siomi teams that a high level of small RNA is produced in the drosophila germ line from transposon repeats in the pericentromeric regions of the chromosomes. We now know that transposon transcripts are processed by a complex Ping-Pong mechanism, which is Dicer independent, and involves the three germ line specific Argonaute proteins, Piwi, Aubergin and Argonaute-3. I will not go in detail in this pathway. Just remind that in Drosophla, the reapeat associated rasiRNA are also piRNAs because they are found associated to these three Argonautes of the PIWI clade. A last word will be to say that piRNAs are though to mediate transponson silencing at the post transcriptional level, through the endonucleolytic activity of the piwi, aub and ago3 proteins. There is a forth small RNA pathway which as been pretty well characterized in pombe and plants by remains elusive in drosophila. Though this pathway, small RNAs arising from transposon repeats direct the methylation of the H3-H9 histone residue and thereby participate to the recruitment of the heterochromatin protein 1 and heterochromatin formation. The proviral activity of the suppressors in flies prompted us to study whether they can perturb one or the other of these known pathways and in particular whether they can perturb chromatin dynamics.

32 Endo-siRNAs silence transposable element in the soma
(as do piRNAs in the germ line) Now, I need to briefly remind you that there is currently 3 distinct small RNA pathways characterized in this model organism. In the microRNA pathway, small stem loop RNA molecule are cropped from primary long RNA by the Rnase Drosha and exported in the cytoplasm by an exportin 5 dependent system. These pre-miRNA are then processed by Dicer-1 and one strand of the miRNA duplex is then loaded into an Argonaute-1 complex which mediates inhibition of translation and destabilization of the target mRNA. Long dsRNA, as I just shown you for viral genomes, are processed by Dicer-2, and loaded as single stranded siRNA into the argonaute-2 RISC complex which mediates an endonucleolytic cleavage of the target mRNA. It has been recently demonstrated by the Hannon and Siomi teams that a high level of small RNA is produced in the drosophila germ line from transposon repeats in the pericentromeric regions of the chromosomes. We now know that transposon transcripts are processed by a complex Ping-Pong mechanism, which is Dicer independent, and involves the three germ line specific Argonaute proteins, Piwi, Aubergin and Argonaute-3. I will not go in detail in this pathway. Just remind that in Drosophla, the reapeat associated rasiRNA are also piRNAs because they are found associated to these three Argonautes of the PIWI clade. A last word will be to say that piRNAs are though to mediate transponson silencing at the post transcriptional level, through the endonucleolytic activity of the piwi, aub and ago3 proteins. There is a forth small RNA pathway which as been pretty well characterized in pombe and plants by remains elusive in drosophila. Though this pathway, small RNAs arising from transposon repeats direct the methylation of the H3-H9 histone residue and thereby participate to the recruitment of the heterochromatin protein 1 and heterochromatin formation. The proviral activity of the suppressors in flies prompted us to study whether they can perturb one or the other of these known pathways and in particular whether they can perturb chromatin dynamics.

33 Transposon repression Heterochromatin formation
Defense against viruses Transposon repression Heterochromatin formation Regulation of gene expression

34 Mathieu Bartoletti Bassam Berry Corinne Besnard-Guerin Anne-Laure Bougé Caroline Jacquier Josette Pidoux Edwige Seguy Hélène Thomassin