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L’ARN interférence

L’ARN interférence. Chantal Vaury GReD UMR/CNRS6247, Clermont Université, INSERM U931 Clermont-Ferrand. Introduction générale. Le principe de conservation. Le principe de conservation. Il existe chez tous les êtres vivants des mécanismes communs tels

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L’ARN interférence

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  1. L’ARN interférence Chantal Vaury GReD UMR/CNRS6247, Clermont Université, INSERM U931 Clermont-Ferrand

  2. Introduction générale Le principe de conservation

  3. Le principe de conservation Il existe chez tous les êtres vivants des mécanismes communs tels que la production d’énergie, la réplication de l’ADN ou le transport des molécules. Il est par conséquent possible d’étudier ces processus en utilisant des organismes modèles faciles à manipuler. Ces processus étant conservés entres les espèces, les résultats obtenus avec le modèle seront transposables à d’autres êtres vivants tels que l’homme. Concernant les eucaryotes, il existe des modèles phares tels que la levure, le nématode, la drosophile et la souris.

  4. Historique de l’ARN interférence

  5. Le nématode: Caenorabditis elegans

  6. Antisense RNA “antisense” experiment Cell 81, 1-20 1995 Molecule Injected % embryonic lethality ZC22 (par-1)antisense 52 ZC22 (par-1)sense 54 H20 0 “Surprisingly, injection of sense ZC22 RNA also induced par-1 phenotypes. It is not clear what accounts for this effect… The basis for the sense effect is under investigation and will not be discussed further…”

  7. RNA polymerases, although highly specific, produce some random or ectopic transcripts Molecules Injected % twitching unc-22 “pure” antisense 0 unc-22 “pure” sense 0 unc-22 sense + antisense 100 Nature 391, 806 1998

  8. RNAi by ingesting dsRNA! - 1998 Timmons and Fire - Nature 295, 854 Bacteria expressing dsRNA GFP-expressing worms…. ….eating regular bacteria ….eating bacteria expressing GFP dsRNA

  9. Screen for RNAi-deficient or rde mutants Tabara et al., 1999 Cell 99, 123 Craig Mello

  10. rde-1 is evolutionarily conserved! Tabara et al., 1999 Cell 99, 123

  11. Attempts to deepen the purple hue of petunias by genetic modification produced unexpected results. Rather than heightening pigmentation, an inserted gene switched colour production off, creating variegated blooms (inset). (Taken from Nature 404, 804 - 808)

  12. Inactivation épigénétique post transcriptionnelle

  13. PTGS and RNAi - a connection? Both are RNA-based gene silencing mechanisms “Posttranscriptional gene silencing occurs in plants and fungi transformed with foreign or endogenous DNA and results in the reduced accumulation of RNA molecules with sequence similarity to the introduced nucleic acid. David Baulcombe Double-stranded RNA induces a similar effect in nematodes, insects, and protozoa.” Hamilton and Baulcombe, 1999. Science 286, 952

  14. Découverte de petits ARN complémentaires des cibles Ces ARN sont appelés small interfering RNA ou siRNA

  15. Small interfering RNA pathways • si RNAs • mi RNAs • pi RNAs • endosi RNAs

  16. Small interfering RNA pathway DICER Cleaves the long dsRNA to make siRNAs RISC (RNA Induced Silencing Complex) Uses the siRNAs as guides to cleave target mRNAs

  17. Le RISC

  18. RNAi pathway Molecular biology central dogma DNA Transcription RNA Translation Protein Cellular function

  19. Des siRNAs aux miRNAs Un monde de petits ARNs régulateurs

  20. miRNA Naturally occuring small RNA Cullen SR, 2005, Nature Genetics, 1163

  21. miRNAi pathway Molecular biology central dogma DNA Transcription RNA Translation Protein Cellular function

  22. microRNAs: “hetero-silencing” * Encoded in genome as hairpin precursors * Processed by DICER into 21-25nt RNAs * Targets loci distinct from the microRNA loci. * Degrade mRNAs with perfect match. * Block translation of mRNAs with imperfect match siRNAs: “auto-silencing” * Agents for RNAi * 21-25nt RNAs processed by DICER (in some animals a different DICER gene than that used for microRNAs) from exo or endogenous dsRNAs * Targets are RNAs from same or very similar locus * Degrade RNAs with perfect match

  23. Des siRNAs et miRNAs aux piRNAs ou rasiRNAs Un monde de petits ARNs régulateurs

  24. Characteristics of Drosophila piRNAs

  25. Drosophila piRNAs Map to Discrete Genomic Loci

  26. The ping-pong model

  27. Représentation schématique d’une région génomique générant un cluster de piRNAs Vagin et al., 2006, Science, 313, 320-324 Carthew, 2006, Science, 313, 305-306.

  28. Endo siRNA NAT: Natural Antisens Transcripts Okamura and Lai, 2008

  29. Exo-siRNA and Endo siRNA

  30. Silencing transcriptionnel en lien avec le silencing post-transcriptionnel

  31. Bloquer l’expression Réduire la stabilité du messager Bloquer sa traduction Empêcher la transcription PTGS ou silencing post-transcriptionnel L’interférence ARN… TGS ou silencing transcriptionnel Structure de la chromatine Immunité intrinsèque et « silencing »

  32. Chromatin packaging into chromosomes

  33. Structure de la chromatine et silencing transcriptionnel

  34. Le code histone Ensemble de modifications chimiques de la chromatine à déchiffrer…

  35. RNAi et silencing transcriptionnel sont parfois liés (S. pombe) Chromatine active Éléments répétés RNAi RITS HMT Chromatine K9met ADN méthyl-transférases H3 HP1/SWI6 HDAC déacetylation H3 H3 Méthylation de l’ADN Chromatine inactive hétérochromatine Les séquences hétérochromatiques répriment leur propre expression. Les séquences hétérochromatiques peuvent réprimer en trans l’expression d’une séquence qui leur est homologue. Volpe TA, et al., 2002, 297, 1833

  36. 6 4 Meth H3 Lysine4 2 0.75 0.5 Meth H3 Lysine 9 0.25 Rdp1- Ago1- wt Dcr1- La voie d’ARN interférence guide le silencing de l’hétérochromatine

  37. URA+ Tandem repeats Silencing en cis La voie d’ARN interférence initie le silencing de l’hétérochromatine RNAi et silencing transcriptionnel sont parfois liés

  38. E E E Propagation d’une structure chromatinienne compacte

  39. Transitivité Ou Comment amplifier le signal?

  40. Unc Unc GFP GFP Targeting GFP abolishes fluorescence but also creates an unexpected, uncoordinated phenotype. This occurs because of the production of double-stranded RNA and consequently small interfering RNAs homologous to the endogenous UNC-22 gene.

  41. Production de trans-siRNAs

  42. Systémie ou comment distribuer le signal

  43. Dispersion de l’ARNi à travers l’organisme A silencing signal moves from the veins into leaf tissue. Green = GFP fluorescence Red = chlorophyll fluorescence that is seen upon silencing of the GFP transgene. C. elegans engineered to express GFP in nuclei. Animals on the right have been treated with a control dsRNA. Animals on the left have been exposed to GFP dsRNA. Some neuronal nuclei remain fluorescent, correlating with low expression of a protein required for systemic RNAi59.

  44. Transitivité et systémie assurent l’efficacité du contrôle par la voie RNAi

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