Molecular Mechanisms of the RNAi/MicroRNA Pathways

 描述（由申请人提供）：RNA干扰（RNAi）和相关途径在全球范围内影响基因组表达，控制各种病理/生理过程，并有望为人类疾病提供新的基于小RNA的治疗方法。RNA诱导沉默复合物（RISC）是RNAi的催化引擎，其中单链siRNA引导Argonaute 2（Ago 2）RNase催化互补mRNA的序列特异性切割。因此，我们将使用多学科的方法来回答以下开放的和根本性的重要问题：1）RISC加载和激活的机制是什么？2)动态RISC汇编的结构基础是什么？目的一是将联合收割机正向遗传筛选和生化分离重组相结合，鉴定新的RNAi因子，确定果蝇RISC装载复合物（RLC）的分子组成，并阐明RLC将双链siRNA从Dicer 2-R2D2复合物转移到Ago 2形成失活的pre-RISC的详细机制。在目标2中，我们将应用一种新的化学网格电子显微镜（EM）技术，我们开发的RISC加载和激活的动态过程的结构基础进行调查。这种尖端技术允许选择性富集在siRNA包被的ChemiC-Grid上组装的重组RLC和RISC，并在接近生理浓度的特定功能状态下对这些复合物进行cryoEM成像。这两个高度互补的目的将提供一个完整的生化/结构框架，了解真核RISC组装的基本机制。这些知识对于理解这些途径被破坏的人类疾病以及开发靶向或利用这些小生物活性RNA的新型疗法至关重要。

 DESCRIPTION (provided by applicant): The RNA interference (RNAi) and related pathways globally impact genome expression, govern diverse patho/physiological processes, and promise novel small RNA-based therapeutics for human diseases. The RNA-induced silencing complex (RISC) is the catalytic engine of RNAi, wherein single-strand siRNA guides Argonaute 2 (Ago2) RNase to catalyze sequence-specific cleavage of complementary mRNA. Thus, we will use a multidisciplinary approach to answer the following open and fundamentally important questions: 1) what are the mechanisms of RISC loading and activation? 2) what are the structural bases of dynamic RISC assembly? In Aim 1, we will combine forward genetic screen and biochemical fractionation & reconstitution to identify new RNAi factors, define the molecular composition of the Drosophila RISC loading complex (RLC), and elucidate the detailed mechanism by which the RLC transfers duplex siRNA from Dicer2-R2D2 complex to Ago2 to form inactive pre-RISC. In Aim 2, we will apply a novel ChemiC-Grid electron microscopy (EM) technology that we developed to investigate structural underpinnings of the dynamic process of RISC loading and activation. This cutting-edge technology allows for selective enrichment of recombinant RLC and RISC assembled on the siRNA-coated ChemiC-Grids and cryoEM imaging of these complexes at specific functional state at near physiological concentrations. These two highly complementary Aims will provide an integrated biochemical/structural framework for understanding the fundamental mechanisms of eukaryotic RISC assembly. The knowledge is essential to understanding human diseases in which these pathways are disrupted, and for developing novel therapeutics that either target or utilize these small bioactive RNAs.