Regulatory Mechanisms of RNA Interferance

描述（由申请人提供）：本研究计划的总体目标是了解RNA干扰（RNAi）的调控机制。RNAi是一种由microRNA （miRNA）和小干扰RNA （siRNA）介导的转录后基因沉默机制。虽然已经确定了许多RNAi成分，但它们在RNAi中的具体功能尚不清楚。迄今为止，只有三种核心成分经过了生化验证。Dicer将长dsRNA和pre-miRNA加工成siRNA和miRNA。Argonaute （Ago）在siRNA或miRNA的引导下，指导序列特异性切割和同源mRNA的翻译抑制。dsrna结合蛋白（dsRBP），如果蝇中的R2D2，促进siRNA从Dicer转移到Ago，形成效应rna诱导沉默（RISC）复合物。

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.