Structure and Mechanism of the RISC-loading Complex

描述（由申请人提供）：微小RNA（miRNA）是一类丰富的22个核苷酸的调控元件，其指导哺乳动物基因的沉默并有助于广泛的细胞过程，包括发育时间、寿命、脑发育、细胞增殖和癌症。miRNA途径中的关键步骤是将miRNA组装成活性形式，即称为RISC的大型核糖核蛋白复合物。该项目旨在表征RISC的组装，重点是了解RISC中miRNA加工和加载的结构细节。研究的重点是一个三蛋白组装称为RISC加载复合物（RLC）。使用生物化学和结构生物学的组合，我们将确定RLC机制的关键特征，可用于治疗目的。生物化学的努力将集中在解剖miRNA识别和加工效率的机制。电子显微镜将用于确定RLC的分子结构，这将指导进一步的生化实验。我们的目标是确定RLC的分子结构停滞在RISC加载的关键步骤，充分可视化这一重要的细胞过程。这些结构和功能相结合的研究将提供全面的机制的见解之一，最基本的，但最不了解的途径控制哺乳动物基因表达。公共卫生相关性：RNA干扰（RNAi）是一种自然过程，通过这种过程，人类基因被关闭或“沉默”，这涉及从寿命到大脑发育到癌症的生物功能。该项目旨在了解决定哪些基因将被RNAi沉默的分子机制。这些结果将有助于将RNAi过程作为治疗人类疾病的强大新型治疗方法的持续努力。

DESCRIPTION (provided by applicant): Micro-RNAs (miRNA) are an abundant class of 22-nucleotide regulatory elements that guide the silencing of mammalian genes and contribute to broad range of cellular processes including developmental timing, life span, brain development, cell proliferation, and cancer. A critical step in the miRNA pathway is the assembly of miRNAs into an active form, a large ribonucleoprotein complex called RISC. This project aims to characterize the assembly of RISC with an emphasis on understanding the structural details underlying processing and loading of miRNAs into RISC. Research is focused on a three-protein assembly termed the RISC-loading complex (RLC). Using a combination of biochemistry and structural biology we will identify key features of the RLC mechanism that may be exploited for therapeutic purposes. Biochemical efforts will focus on dissecting the mechanisms underlying miRNA recognition and processing efficiency. Electron microscopy will be used to determine the molecular architecture of the RLC, which will guide further biochemical experiments. We aim to determine molecular structures of the RLC stalled at key steps in RISC-loading to fully visualize this essential cellular process. These combined structural and functional studies will provide comprehensive mechanistic insights into one of the most fundamental but least understood pathways of controlling mammalian gene expression. PUBLIC HEALTH RELEVANCE: RNA interference (RNAi) is a natural process by which human genes are turned off, or "silenced", which is involved in biological functions ranging from life span, to brain development, to cancer. This project aims to understand the molecular machinery that decides which genes will be silenced by RNAi. Results will contribute to on-going efforts to harness the RNAi process as a powerful new type of therapeutic for the treatment of human disease.