MECHANISMS OF DSRNA-INDUCED GENE SILENCING

在进化上多样化的生物群体中，包括线虫、果蝇、锥虫、舟形虫、多头蛇、斑马鱼、植物和真菌，双链RNA的引入以序列特异性的方式诱导基因沉默。这一过程被称为RNA干扰(RNAi)、转录后基因沉默(PTGS)和抑制。虽然dsRNA诱导的基因沉默可能发挥重要的生物学作用，但人们对这一现象的普遍兴趣在一定程度上是因为它作为一种研究工具的实用性。尽管DERNA诱导的基因沉默具有明显的重要性，但这种现象背后的机制仍然不清楚。我们已经采取了生物化学的方法来破译dsRNA抑制基因表达的机制。我们已经证明，当导入培养的果蝇S2细胞时，dsRNA通过降低与dsRNA同源的mRNAs的水平来抑制基因表达。转基因细胞的提取物含有一种核酸酶，可以降解同源的合成RNA，但对异源RNA没有活性。我们已经证明了这种核酸酶是一种RNP，并已经鉴定出一种可能指导底物选择的RNA成分。我们将这种酶命名为RISC(RNA诱导沉默复合体)。在这一应用中，我们概述了我们通过体外和体内研究相结合的方法来阐明dsRNA诱导基因沉默的潜在机制。

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.