Structural Basis for RNA Silencing by Human Argonaute2

描述（由申请人提供）：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，干细胞和生殖系维持以及癌症进展。在分子水平上，所有的RNA沉默途径，如microRNA （miRNA）调控途径，都是由一个名为Argonaute的RNA结合蛋白家族介导的。Argonaute蛋白具有独特的结合小调控rna的能力，并利用编码的序列信息来定位和沉默互补的靶rna。RNA沉默的多功能性和强大性源于Argonaute可以装载任何序列的小RNA，因此可以编程以任何RNA为目标进行沉默。然而，尽管阿尔戈瑙特在人类生物学中的重要性和尚未开发的治疗潜力，但缺乏对阿尔戈瑙特的详细结构理解。事实上，有关Argonaute的结构及其功能的信息是有限的，这些信息要么来自遥远的细菌形式的酶，它们使用DNA引导分子，要么来自真核生物形式的孤立区域。该研究的贡献将提供全长人类Argonaute2 （Ago2）高分辨率结构和功能分析。本研究的总体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)必要辅助因子TNRC6的结合。在前期工作中，Ago2的结构已经确定，并将作为本文提出的研究的启动平台。在Aim 1中，将使用结构和生化方法相结合的方法来确定Ago2如何结合引导rna并定位它们以有效地识别目标rna。这项工作将为合理设计改进的sirna提供结构见解。在Aim 2中，将采用类似的方法来理解Ago2识别靶rna的机制。这些研究将为其他实验室开发的经验miRNA靶向“规则”提供结构基础，并为研究和治疗目的操纵这些rna开辟新的途径。在Aim 3中，将确定Ago2与TNRC6关联的结构基础。这些结果将定义指导RNA在体内沉默所需的大量复合物组装的一般原则。这些结构和功能研究结合起来，将为人类RNA沉默的最基本但鲜为人知的组成部分之一提供全面的机制见解。

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.