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.
