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Hannon, Nature 418:244-251 Jacques et al, Nature 418:435-8 Carmichael Nature 418:379-380 Allshire, Science 297:1818-9
Why RNAi?
Hypotheses and Clues Include: RNAi mechanism evolved to immobilize transposable elements and silence RNA viruses ie Mut7 -/- C. elegans; has a mutator phenotype b/c transposable element Later RNAi important in silencing chromatin may recruit Clr4 histone H3 methylase small RNAs have been correlated w/ methylation of promoter DNA of Arabidopsis (S.pombe has no DNA methylation) both siRNAs and miRNAs regulate gene expression
Introduced 22 nucleotide synthetic siRNAs (complementary to HIV target +/- GFP) into human cell lines/ primary lymphocytes RESULTS:
DO NOT ACTIVATE PKR PATHWAY and siRNAs SPECIFICALLY DEGRADE HIV-1 mRNA, dsRNA-activated protein kinase
Heterochromatin*-repetitive, condensed part of genome Post-translation modific. of histone tails important Transgenes inserted into heterochromatin are shut off
in S. pombe repetitive DNA near centromeres is silenced via METHYLATION of H3 Lys9 and binding of Swi6 (gene express ON if Lys4 methylated)
Volpe et al. found that deleting genes in RNAi pathway (argonaute, Dicer, Rdp1*) lead to LOSS of GENE SILENCING of transgenes inserted into heterochromatin RNAi and Heterochromatin Silencing are RELATED Pathways
How does the RNAi machinery aid in the formation of silent chromatin?
Possibility that siRNAs bring methyltransferases to the target loci, where they are important in histone tail modification
ie. Drosoph. targets acteyltransferase w/ RNA binding chromodomain to histone H4
Mouse X inactivation and Igf2r imprinting are mediated by noncoding antisense RNA
Possibly in organisms w/ DNA methylation; Histone protein modification similar to S. pombe would in turn cause DNA methylation and subsequent gene silencing regulation
FOR MORE INFO. ON CORRELATION SEE Volpe et al. SCI 297:1833-1837