The role of Dicer in siRNA loading and RISC assembly

RNA干扰(RNAi)是由双链(DS)RNA分子触发的，已发展成为一种广泛使用的实验室工具。此外，第一批基于RNAi的药物正在开发中，并已达到临床第三阶段水平。RNAi由短干扰RNAs(SiRNAs)引导，siRNAs主动整合到RNA诱导的沉默复合体(RISC)中。较长的dsRNAs被RNaseIII酶处理成siRNAs。ArgAert(AGO)蛋白家族的一个成员形成了RISC中siRNAs的直接蛋白质结合伙伴。虽然RNAi的基本机制已经被很好地理解，但在哺乳动物细胞中，将siRNAs装载到RISC中仍然是难以捉摸的。已有研究表明，DICER与AGO蛋白稳定地相互作用。然而，迪格尔对RISC加载的贡献尚不清楚。在初步实验中，我们已经证明了在Dird缺陷细胞中，RISC负载受到了强烈的损害。此外，异位表达的迪切尔可以挽救这种效应。因此，这项建议旨在详细描述DICER在RISC加载中的作用。利用质谱学的方法，我们还将研究DICER对RISC蛋白质组成的影响。我们的项目不仅将阐明RNAi的原理机制，而且还可能有助于从总体上改进RNAi的使用。

RNA interference (RNAi) is triggered by double stranded (ds) RNA molecules and has developed into a widely used lab tool. Moreover, the first RNAi-based drugs are being developed and have already reached clinical phase III levels. RNAi is guided by short interfering RNAs (siRNAs), which are actively incorporated into the RNA-induced silencing complex (RISC). Longer dsRNAs are processed to siRNAs by the RNase III enzyme Dicer. A member of the Argonaut (Ago) protein family forms the direct protein-binding partner for siRNAs within RISC. Although the basic mechanisms of RNAi are reasonably well understood, loading of siRNAs into RISC remains elusive in mammalian cells. It has been shown that Dicer stably interacts with Ago proteins. The contribution of Dicer to RISC loading, however, remains unknown. In preliminary experiments we have demonstrated that RISC loading is strongly impaired in Dicer deficient cells. Furthermore, ectopically expressed Dicer can rescue this effect. Therefore, this proposal aims at a detailed characterization of the role of Dicer in RISC loading. Using mass spectrometry, we will also investigate the effect of Dicer on the protein composition of RISC. Our project will not only elucidate principle RNAi mechanisms but may also help to improve the use of RNAi in general.