MECHANISMS OF DSRNA-INDUCED GENE SILENCING

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In an evolutionarily diverse group of organisms that includes C. elegans, Drosophila, trypanosomes, planaria, hydra, zebrafish, plants and fungi, introduction of double-stranded RNA induces gene silencing in a sequence-specific fashion. This process has been termed variously RNA interference (RNAi), PTGS (post-transcriptional gene silencing) and quelling. Although dsRNA-induced gene silencing may play important biological roles, general interest in this phenomenon has been fueled, in part, by its utility as a research tool. Despite the obvious importance of deRNA-induced gene silencing, the mechanisms underlying this phenomenon have remained obscure. We have taken a bio chemical approach toward deciphering the mechanisms by which dsRNA suppresses gene expression. We have shown that, upon transfection into cultured, Drosophila S2 cells, dsRNA inhibits gene expression by reducing the levels of mRNAs that are homologous to the dsRNA.. Extracts of transfected cells contain a nuclease that degrades cognate, synthetic mRNAs but that is inactive against heterologous RNAs. We have demonstrated that this nuclease is an RNP and have identified an RNA component that may guide substrate selection. We have designated this enzyme RISC (RNA-induced silencing complex). In this application, we outline our approach toward elucidating the mechanisms underlying dsRNA-induced gene silencing through a combination of in vitro and in vivo studies.