Rna silencing

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Rna silencing

  1. 1. RNA Silencing/RNAi
  2. 2. RNA SILENCING• The process of knock-down of any gene• Functions as a antiviral mechanism• May be post transcriptional as well as transcriptional gene silencing• Two types of short RNA molecules are involved  miRNA  siRNA
  3. 3. Short history of post-transcriptional gene silencing 1990 Jorgensen : Introduction of transgenes homologous to endogenous genes often resulted in plants with both genes suppressed! Called Co-suppression Resulted in degradation of the endogenous and the transgene mRNA
  4. 4.  DiscoveryInhibition by injectedanti-sense RNASense RNA had similar Jorgensen et al., Nature (1988)effects! Napoli, Lemieux, Jorgensen. Plant Cell (1990)
  5. 5. Amin, I and K. Hussain 2011
  6. 6. Contd….1995 Guo and Kemphues: -injection of either antisense or sense RNAs in the germline of C. elegans was equally effective at silencing homologous target genes1998 Mello and Fire: -extension of above experiments, combination of sense and antisense RNA (= dsRNA) was 10 times more effective than single strand RNA
  7. 7. Source for siRNA:EndogenousExogenous
  8. 8. double-stranded RNAs are produced by:– transcription of inverted repeats– viral replication– transcription of RNA by RNA-dependent RNA- polymerases (RdRP) double-stranded RNA triggers cleavage of homologous mRNA PTGS-defective plants are more sensitive to infection by RNA viruses in RNAi defective nematodes, transposons are much more active
  9. 9. Dicer Double-stranded RNA triggers processed into siRNAs by enzyme RNAseIII family, specifically the Dicer family Dicer family proteins are ATP-dependent nucleases. These proteins dice long dsRNA into small RNAduplexes of 21-26nt sizes. Loss of dicer: loss of silencing process
  10. 10. Citwood and Timmermans, Nature 2010
  11. 11. RISC• RISC has helicase, exonuclease, endonucelase and homology searching proteins.• Initial RISC is inactive until transformed into active form by unwinding of the siRNA duplex and loss of sense (passenger) strand• Antisense (guide) strand defines specificity of RNAi
  12. 12. Spreading of silencing and amplificationvia RdRP (RNA-dependent RNA polymerase) S. pombe, plants, worms systemic silencing; amplification of silencingNot in Drosophila or in humans
  13. 13. miRNA siRNAnoncoding RNA gene repeat associatedPolII transcribed PolII/V transcribed or bidirectional transcriptionHairpin (loop, mismatches) double stranded RNAPTGS PTGSTGS? TGS
  14. 14. Biogenesis of microRNACullen Nature Immunology 7:563 2006
  15. 15. Translational Repression
  16. 16.  Functions --- RNA-Directed De novo Methylation (RdDM) DCL3, 24 nt siRNAs, AGO4, MET1/DRM2 AGO4 Marjori A. Matzke & James A. Birchler Nature Reviews Genetics 6, 24-35 (2005)
  17. 17. Why is RNAi important?begun in worms, flies, and plants - as an accidentalobservation.general applications in mammalian cells.probably much more common than appreciatedbefore: – it was recently discovered that small RNAs correspond to centromer heterochromatin repeats – RNAi regulates heterochromatic silencingFaster identification of gene function
  18. 18. Most widely held view is that RNAi evolved to protect the genome from viruses (or other invading DNAs or RNAs)Recently, very small (micro) RNAs have been discovered in several eukaryotes that regulate developmentally other large RNAs – May be a new use for the RNAi mechanism besides defense
  19. 19. Contd….. Powerful for analyzing unknown genes in sequence genomes.  efforts are being undertaken to target every human gene via miRNAs Gene therapy: down-regulation of certain genes/mutated alleles Cancer treatments – knock-out of genes required for cell proliferation – knock-out of genes encoding key structural proteins

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