Structure and Mechanism of the RISC-loading Complex

 描述(申请人提供)：microRNAs(MiRNAs)是一类丰富的小(~22个核苷酸)调控RNA，控制着人类超过一半的蛋白质编码基因的表达，是各种哺乳动物生理过程中必不可少的组成部分。许多miRNAs有助于细胞内环境的稳定，而miRNA的异常表达往往与细胞生长失调和癌症有关。尽管miRNAs在人类生物学中具有广泛的功能，但在理解miRNA生物发生中确保保真度的分子机制方面仍存在根本差距。这些差距的持续存在代表着一个重大的医学问题，因为在它被填补之前，对促进肿瘤生长的miRNA生物发生因子中的遗传异常的理解在很大程度上仍然是不可理解的。长期目标是了解miRNA途径和相关的RNA沉默过程，以提供可用于人类疾病诊断和治疗的详细见解。本应用的目的是剖析人类RISC负载复合体(RLC)的机制，RLC是一个大的分子组装，催化miRNA生物发生的最后步骤，并确定哪些细胞基因将被miRNA途径下调。拟议的工作将探索这样一种假设，即与癌症相关的RLC组件中的基因突变是通过扰乱miRNA生物发生的机制来发挥作用的，从而导致miRNA表达异常，从而推动肿瘤的发生。提出这项研究的理由是，对RLC结构和机制的详细了解将为各种人类肿瘤的生物学提供分子洞察力。在强大的初步数据的指导下，这一目标将通过追求两个具体目标来实现：1)确定RISC负载复合体的结构；2)剖析RISC负载的机制和指导RNA选择。在第一个目标下，将使用X射线结晶学和电子显微镜来确定人类RLC及其单个成分的分子结构。这些努力将通过电子显微镜检查小颗粒和现有晶体的创新方法来促进。完成的结果将大大推进RNA沉默领域，因为目前唯一发表的RLC结构已被证明是不正确的，复杂组装的细节在文献中变得混乱和有争议。在第二个目标下，野生型和突变型RLCs中的离散RISC加载步骤将使用已建立的生化分析方法进行检查。这一目标具有重要意义，因为它将为RISC加载过程提供机械细节(目前仅在经验水平上了解)，以及对驱动肿瘤发生的突变如何扰乱miRNA生物发生机制的直接和具体的见解。因此，这些结果将有助于了解和治疗各种形式的癌症。整个提案是创新的，因为它确定并解决了特定的临床对生物学洞察力的紧迫性，可以通过经过验证的结构和生化方法来缓解这一点。

 DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are an abundant class of small (~22 nt) regulatory RNAs that control the expression of more than half of all protein-coding genes in humans and are essential components in diverse mammalian physiological processes. Many miRNAs contribute to cellular homeostasis and aberrant miRNA expression is often associated with dysregulated cell growth and cancer. Despite the widespread function of miRNAs in human biology there are fundamental gaps in understanding the molecular mechanisms ensuring fidelity in miRNA biogenesis. Continued existence of these gaps represents a significant medical problem because, until it is filled, understanding of genetic abnormalities in miRNA biogenesis factors that promote tumor growth will remain largely incomprehensible. The long-term goal is to understand the miRNA pathway and related RNA silencing processes to provide detailed insights that can be harnessed for the diagnosis and treatment of human disease. The objective of this application is to dissect the mechanism of the human RISC-loading complex (RLC), which is a large molecular assembly that catalyzes the final steps of miRNA biogenesis and determines which cellular genes are to be down-regulated by the miRNA pathway. The proposed work will explore the hypothesis that genetic mutations in RLC components associated with cancer act by perturbing the mechanism of miRNA biogenesis, leading to aberrant miRNA expression, which in turn drives tumorigenesis. The rational for the proposed research is that detailed understanding of the structure and mechanism of the RLC will provide molecular insights into the biology of diverse human tumors. Guided by strong preliminary data, this objective will be achieved by pursuing two specific aims: 1) Determine structures of the RISC-loading Complex; and 2) Dissect the mechanisms of RISC-loading and guide RNA selection. Under the first aim, X-ray crystallography and electron microscopy will be used to determine molecular structures of the human RLC and its individual components. These efforts will be facilitated by innovative methods for examining small particles by electron microscopy and crystals that are already in hand. Completed results will significantly advance the RNA silencing field because, currently, the only published RLC structure has been shown to be incorrect and the details of how the complex assembles have become muddled and contested in the literature. Under the second aim, discrete RISC-loading steps in wild-type and mutant RLCs will be examined using established biochemical assays. This aim is significant because it will provide mechanistic details into the process of RISC- loading, which is currently only understood at an empirical level, as well as direct and specific insights into how mutations the drive tumorigenesis perturb the mechanisms in miRNA biogenesis. These results will thereby inform efforts to understand and treat diverse forms of cancer. The overall proposal is innovative because it identifies and addresses a specific clinical exigency for biological insight that can be mitigated through proven structural and biochemical approaches.