MECHANISMS OF DSRNA-INDUCED GENE SILENCING

在一个进化上多样化的生物群体中，包括C。线虫、果蝇、锥虫、涡虫、水螅、斑马鱼、植物和真菌中，双链RNA的引入以序列特异性方式诱导基因沉默。 这个过程被称为RNA干扰（RNAi），PTGS（转录后基因沉默）和镇压。 尽管dsRNA诱导的基因沉默可能发挥重要的生物学作用，但部分由于其作为研究工具的实用性，对这种现象的普遍兴趣已经得到了推动。 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。 我们已经表明，在转染到培养的果蝇S2细胞中后，dsRNA通过降低与dsRNA同源的mRNA的水平来抑制基因表达。转染细胞的提取物含有降解同源合成mRNA但对异源RNA无活性的核酸酶。 我们已经证明，这种核酸酶是一种RNP，并确定了一种RNA组分，可以指导底物的选择。 我们将这种酶命名为RISC（RNA诱导的沉默复合物）。 在这个应用程序中，我们概述了我们的方法，通过体外和体内研究相结合，阐明dsRNA诱导的基因沉默的机制。

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.