Regulatory Mechanisms of RNA Interferance

描述（由申请人提供）：本研究提案的总体目标是了解RNA干扰（RNAi）的调控机制。RNAi是由microRNA（miRNA）和小干扰RNA（siRNA）介导的一种转录后基因沉默机制。虽然已经鉴定了许多RNAi组分，但它们在RNAi中的具体功能仍不清楚。迄今为止，只有三种核心成分经过生物化学验证。Dicer将长dsRNA和pre-miRNA加工成siRNA和miRNA。Argonaute（Ago）由siRNA或miRNA引导，指导序列特异性切割和翻译抑制同源mRNA。双链RNA结合蛋白（dsRBP），如果蝇中的R2D2，能促进siRNA从Dicer转移到Ago，形成效应子RNA诱导沉默（RISC）复合物。 生物化学分离和重组是深入了解RNAi分子机制的重要途径。在本研究中，我们建议使用重组Dicer/dsRBP/Ago蛋白来重建holo-RISC活性。我们的重组系统将使我们处于一个独特的位置，以解决该领域的几个基本和长期存在的问题：什么因素构成全息RISC？RISC是如何组装的？RNAi是如何调控的？为了实现我们的目标，我们设计了三个具体的研究途径。在目的1中，我们使用纯化的重组Dcr- 2/R2D2/Ago 2蛋白重建了果蝇dsRNA启动的RISC活性。这种重组系统将使我们能够深入研究RISC组装的生化机制。目的二是利用遗传学和生物化学相结合的方法，鉴定果蝇RNAi新的调控因子，并研究RNAi调控的生物化学机制。在目标3中，我们将使用重组Dicer/dsRBP/Ago 2蛋白重建人holo-RISC活性，并研究通过dsRBP的磷酸化调节人RNAi。RNAi和相关途径作为控制基因组活动的基本和全球机制出现。对RNAi调控机制的研究将把该领域与更广泛的生物学背景联系起来，并为小分子调控RNA在生物学和疾病中的正常和病理调控提供生化基础。另一方面，对RNAi机制的理解将有助于合理设计新的和改进的基因沉默技术。RNAi沉默技术的应用将对广泛的生物医学科学产生重大影响，从了解基础生物学，揭示人类疾病的分子基础，到开发新的治疗方法。

DESCRIPTION (provided by applicant): The overall goal of this research proposal is to understand the regulatory mechanisms of RNA interference (RNAi). RNAi is a post-transcriptional gene-silencing mechanism mediated by microRNA (miRNA) and small interfering RNA (siRNA). Although a number of RNAi components have been identified, their specific functions in RNAi remain unclear. To date, only three core components are biochemically validated. Dicer processes long dsRNA and pre-miRNA to siRNA and miRNA. Argonaute (Ago), guided by a siRNA or miRNA, directs sequence-specific cleavage and translational repression cognate mRNA. DsRNA-binding protein (dsRBP), such as R2D2 in flies, facilitates siRNA transfer from Dicer to Ago to form the effector RNA-induced silencing (RISC) complex. Biochemical fractionation and reconstitution is a powerful and essential approach to understand the in-depth molecular mechanisms of RNAi. In the current study, we propose to reconstitute the holo-RISC activities by using recombinant Dicer/dsRBP/Ago proteins. Our reconstitution system will place us in a unique position to address several fundamental and long standing questions in the field: What factors constitute holo-RISC? How is RISC assembled? How is RNAi regulated? To achieve our objectives, we have devised three specific avenues of research. In Aim 1, we have reconstituted Drosophila dsRNA-initiated RISC activity using purified recombinant Dcr- 2/R2D2/Ago2 proteins. This reconstitution system will allow us to investigate the in-depth biochemical mechanisms of RISC assembly. In Aim 2, we will employ a combinatory genetic and biochemical approach to identify novel regulators of Drosophila RNAi and study the biochemical mechanisms of RNAi regulation. In Aim 3, we will reconstitute human holo-RISC activity using recombinant Dicer/dsRBP/Ago2 proteins and investigate regulation of human RNAi by phosphorylation of dsRBP. The RNAi and related pathways emerge as a fundamental and global mechanism to control genome activities. Studies of the regulatory mechanisms of RNAi will connect the field to a wider biological context and provide the biochemical basis for normal and pathological regulations of small regulatory RNAs in biology and disease. On the other hand, the mechanistic understanding of RNAi will facilitate rational design of new and improved gene-silencing technologies. Applications of RNAi silencing technologies will have a significant impact on a broad spectrum of biomedical sciences, ranging from understanding the basic biology, uncovering the molecular basis of human disease, to developing novel therapeutics.