Structure and Mechanism of the RISC-loading Complex

 描述（由申请人提供）：微小RNA（miRNA）是一类丰富的小（~22 nt）调控RNA，其控制人类中超过一半的所有蛋白质编码基因的表达，并且是多种哺乳动物生理过程中的必需组分。许多miRNA有助于细胞内稳态，并且异常的miRNA表达通常与失调的细胞生长和癌症相关。尽管miRNA在人类生物学中具有广泛的功能，但在理解确保miRNA生物发生中的保真度的分子机制方面存在根本性的差距。这些缺口的持续存在代表了一个重大的医学问题，因为在填补缺口之前，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通路和相关的RNA沉默过程，以提供可用于诊断和治疗人类疾病的详细见解。本申请的目的是剖析人RISC加载复合物（RLC）的机制，RLC是催化miRNA生物合成的最后步骤并确定哪些细胞基因将被miRNA途径下调的大分子组装。拟议的工作将探索与癌症相关的RLC组分中的基因突变通过扰乱miRNA生物发生机制而起作用的假设，导致异常的miRNA表达，从而驱动肿瘤发生。提出研究的理由是，详细了解RLC的结构和机制将为不同人类肿瘤的生物学提供分子见解。在强有力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加载复合物的结构;以及2）剖析RISC加载和指导RNA选择的机制。在第一个目标下，X射线晶体学和电子显微镜将用于确定人类RLC及其单个组分的分子结构。这些努力将通过电子显微镜检查小颗粒的创新方法和已经掌握的晶体来促进。完成的结果将显著推进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，并且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，将使用已建立的生物化学测定来检查野生型和突变型RLC中的离散RISC加载步骤。这一目标是重要的，因为它将提供RISC加载过程的机制细节，这是目前仅在经验水平上理解的，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，这些结果将为理解和治疗不同形式的癌症提供信息。总体建议是创新的，因为它确定并解决了一个特定的临床迫切需要的生物学洞察力，可以通过证明结构和生化方法减轻。

 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.