The role of Dicer in siRNA loading and RISC assembly

RNA干扰（RNA interference, RNAi）是由双链RNA分子触发的，目前已成为广泛应用的实验室工具。此外，第一批基于rnai的药物正在开发中，并已达到临床III期水平。RNAi由短干扰rna （sirna）引导，sirna被主动结合到rna诱导的沉默复合体（RISC）中。较长的dsRNAs被RNase III酶Dicer加工成sirna。Argonaut （Ago）蛋白家族的一个成员在RISC中形成sirna的直接蛋白结合伙伴。尽管RNAi的基本机制已经被很好地理解，但在哺乳动物细胞中将sirna装载到RISC中仍然是难以捉摸的。已经证明Dicer与Ago蛋白稳定地相互作用。然而，Dicer对RISC加载的贡献仍然未知。在初步实验中，我们已经证明，在Dicer缺陷细胞中，RISC负载严重受损。此外，异位表达的Dicer可以挽救这种作用。因此，本提案旨在详细描述Dicer在RISC加载中的作用。利用质谱法，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，而且可能有助于提高RNAi的总体使用。

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.