Structural Basis for RNA Silencing by Human Argonaute2

描述(申请人提供)：RNA沉默是指一组广泛存在的基因调控途径，深深植根于人类生物学的几乎每个方面，包括大脑发育、干细胞和生殖细胞维持以及癌症进展。在分子水平上，所有的RNA沉默途径，如microRNA(MiRNA)调控途径，都是由一个专门的RNA结合蛋白家族ArgAerte介导的。ArgAerte蛋白是唯一能够结合小调节RNA并使用编码的序列信息来定位和沉默互补的靶RNA的蛋白质。RNA沉默的多功能性和威力源于这样一个事实，即ArgAerte可以装载任何序列的小RNA，从而可以被编程为靶向任何RNA以进行沉默。然而，尽管ArgAerte在人类生物学中很重要，而且到目前为止还没有开发出治疗潜力，但对ArgAerte的结构缺乏详细的了解。事实上，将ArgAerte的结构与其功能联系起来的信息是有限的，要么来自使用DNA引导分子的遥远的细菌形式的酶，要么来自真核形式的孤立结构域。这项拟议研究的贡献将为全长人类Argavite2(Ago2)提供高分辨率的结构和功能分析。这一建议的首要目标是在详细的结构和机制水平上了解Ago2的三个关键相互作用：(1)引导RNA结合；(2)靶RNA识别；(3)必要的辅助因子TNRC6的结合。在初步工作中，Ago2的结构已经确定，并将作为这里提出的研究的启动平台。在目标1中，将使用结构和生化方法的组合来确定Ago2如何与引导RNA结合并定位它们，以有效地识别靶RNA。这项工作将为改进的siRNA的合理设计提供结构洞察力。在目标2中，将采取类似的方法来理解Ago2识别靶RNA的机制。这些研究将为其他实验室开发的经验性miRNA靶向“规则”提供结构基础，并为出于研究和治疗目的操纵这些RNA开辟新的途径。在目标3中，将确定AGO2与TNRC6结合的结构基础。这些结果将确定指导体内RNA沉默所需的大量复合体组装的一般原则。结合起来，这些结构和功能研究将为人类RNA沉默最基本但知之甚少的组成部分之一提供全面的机制洞察。

DESCRIPTION (provided by applicant): RNA silencing refers to a group of widespread gene-regulatory pathways deeply rooted in nearly every facet of human biology, including brain development, stem-cell and germ-line maintenance and cancer progression. At the molecular level, all RNA silencing pathways, such as the microRNA (miRNA) regulatory pathway, are mediated by a specialized family of RNA-binding proteins named Argonaute. Argonaute proteins are uniquely capable of binding small regulatory RNAs and using the encoded sequence information to locate and silence complementary target RNAs. The versatility and power of RNA silencing arises from the fact that Argonaute can be loaded with a small RNA of any sequence and thus can be programmed to target any RNA for silencing. However, despite the importance of Argonaute in human biology and the as-of-yet untapped therapeutic potential, a detailed structural understanding of Argonaute is lacking. Indeed, information relating the structure of Argonaute to its functions is limited, derived either from distant, bacterial forms of the enzyme, which employ DNA guide molecules, or from isolated domains of eukaryotic forms. The contribution of the proposed research will provide high- resolution structures and functional analyses of full-length human Argonaute2 (Ago2). The overarching goal of this proposal is to understand three key interactions of Ago2 on a detailed structural and mechanistic level: (1) guide RNA binding; (2) target RNA recognition; and (3) the binding of the necessary accessory factor TNRC6. In preliminary work, the structure of Ago2 has already been determined and will serve as the launching platform for the studies proposed here. In Aim 1, a combination of structural and biochemical approaches will be used to determine how Ago2 binds guide RNAs and positions them for efficiently identifying target RNAs. This work will provide structural insights for the rational design of improved siRNAs. In Aim 2, a similar approach will be taken to understand the mechanism by which Ago2 recognizes target RNAs. These studies will provide a structural basis for the empirical miRNA targeting "rules" developed by other labs and open new avenues for manipulating these RNAs for both research and therapeutic purposes. In Aim 3, the structural basis for the association of Ago2 with TNRC6 will be determined. These results will define the general principles that guide the assembly of the massive complexes required for RNA silencing in vivo. Combined, these structural and functional studies will provide comprehensive mechanistic insights into one of the most fundamental-but poorly understood-components of RNA silencing in humans.