Structure and Mechanism of the RISC-loading Complex

项目摘要/摘要 Micro-RNAs(MiRNA)是一类丰富的22核苷酸调控元件，它 引导哺乳动物基因的沉默，并有助于广泛的细胞 包括发育时间、寿命、大脑发育、细胞 增殖和癌症。MiRNA途径中的一个关键步骤是组装 MiRNAs变成一种活性形式，一种被称为RISC的大型核糖核蛋白复合体。这个项目 旨在描述RISC的装配特征，重点是了解 MiRNAs处理和加载到RISC的结构细节。研究是 重点研究了一种名为RISC负载复合体(RLC)的三蛋白质组装。使用 结合生物化学和结构生物学，我们将确定关键特征 可用于治疗目的的RLC机制。生化方面的努力将 重点剖析miRNA识别和处理的潜在机制 效率。电子显微镜将用来确定分子的结构 RLC，这将指导进一步的生化实验。我们的目标是确定 RLC的分子结构在RISC加载的关键步骤中停滞不前，以充分显示这一点 基本的细胞过程。这些结构和功能相结合的研究将提供 对最基本但最不重要的问题之一的全面机械洞察 了解控制哺乳动物基因表达的途径。项目叙事 RNA干扰(RNAi)是人类基因关闭或关闭的自然过程 “沉默”，它涉及从寿命到大脑的各种生物功能 发展，到癌症。这个项目的目的是了解 决定哪些基因将被RNAi沉默。结果将有助于持续的努力 利用RNAi过程作为一种有效的新型治疗方法 人类疾病。

Project Summary/Abstract 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. Project Narrative 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.