RNA诱导沉默复合物的动态特性及其对靶mRNA识别与切割机制研究

Argonaute proteins emerged early on as key players within a complex machinery that exists in all RNAi-competent cells. They interact with microRNA molecules to form an enzymatically active complex called RNA-induced silencing complex (RISC). The RISC complex silences target genes by binding to messenger RNA that has sequence complementarity to the Argonaute-bound RNA. Biochemical and crystallographic studies of argonaute proteins and RISC complex have offered exciting insights into the molecular mechanisms that are central to RNA silencing pathways. However, the structural dynamics of RISC and mechanism of target selection in RNA silencing remain unrevealed. In this application, we plan to develop new strategies to study the structural dynamics of argonaute protein in RISC and the mechanism of molecular recognition and cleavage of target mRNA by RISC by combining use of single-molecule techniques, molecular dynamics simulations and bioinformatics. Firstly, the structural dynamics of argonaute protein during the recognition and cleaving events will be studied by single-molecule FRET technique and molecular dynamics simulations. Secondly, the binding affinity and selectivity of target mRNA by RISC will be studied by using single-molecule manipulation techniques and bioinformatics analysis. Finally, the molecular mechanism of whole process of target selection, binding and cleavage of mRNA by RISC will be revealed.

Argonaute蛋白是miRNA基因沉默通路中的关键蛋白因子，它通过与miRNA引导链形成RNA诱导基因沉默复合物(RISC)，实现对靶mRNA表达的调控。有关RISC的结构生物学研究已经取得了诸多突破性进展，然而其结构本身的动态特性及其与靶mRNA的识别机理研究仍有待深入。本项目拟以RISC为研究对象，首先通过合理设计对Argonaute蛋白或RNA进行荧光标记，利用单分子荧光技术研究RISC与靶mRNA识别过程中Argonaute蛋白的动态行为，并借助分子动力学模拟方法，初步建立Argonaute蛋白的动态模型。随后利用单分子操纵技术，对RISC与不同序列或结构的靶mRNA的识别特异性及切割作用强弱进行系统研究，并利用生物信息学方法进行分析，了解RISC复合物对靶mRNA识别选择性的结构基础，阐明RISC对mRNA识别与切割过程的分子机制，为新型小干扰RNA的设计提供指导。

RNA诱导沉默复合物（RNA-induced silencing complex, RISC）是所有由小RNA介导的特异基因沉默作用过程的关键效应元件，其核心成分是小RNA向导链和Argonaute家族蛋白(AGO蛋白)。RISC的动态特性以及其对靶 mRNA 的识别机理仍不十分清楚。本项目以 嗜热菌RISC为研究对象，利用原子力显微镜单分子力谱技术，研究了RISC 对不同序列的靶 DNA的识别强度及特异性。研究发现AGO蛋白在RISC与靶DNA识别过程中起着重要的调控作用：由于AGO的存在，向导链DNA的种子区域呈较为稳定的A-构型，其对靶DNA的结合大大提高；另一方面，由于种子区域的DNA构象被AGO所限制，使RISC在靶DNA上的滑行更加容易，表现在stretching模式下，RISC与靶DNA的解离力比DNA本身要小。这一发现为阐明RISC对靶RNA的搜索以及检查机制提供了新的思路。