Biochemical Studies of Drosophila RNA-induced Silencing Complex

描述(由申请人提供)： 此应用程序解决广泛的挑战领域(06)使能技术，主题06-GM-105小RNA。RNA干扰(RNAi)是一种由小干扰RNA(SiRNA)或微小RNA(MiRNA)介导的转录后基因沉默机制。RNAi在后生动物中的广泛存在以及在生物学和疾病中的基础作用，突显了RNAi的重要性。RNAi的催化引擎是RNA诱导沉默复合体(RISC)，它指导靶mRNA的序列特异性切割。RISC的核糖核酸酶Ago2与单链siRNA之间可以形成最小的RISC，但与双链siRNA之间不能形成RISC。在果蝇中，Dicer2和R2D2协调地将新生双链siRNA招募到Ago2，以促进RISC组装。全息RISC的重建是RNAi领域的一大挑战。这项建议的目标是用生化分离和重组的方法来理解果蝇RISC。在目标1中，我们用纯化的重组Dicer2/R2D2/Ago2蛋白重组了dsRNA和双链siRNA启动的RISC活性。此外，该核心RISC重组系统被用于纯化一种新的由Translin和Trax蛋白组成的RNAi激活剂。我们将了解Translin/Trax复合体促进果蝇RISC组装和活性的机制。在目标2中，我们发现RISC负载复合体(RLC)除了含有Dicer2/R2D2和siRNA外，还含有未知因子(S)，它的形成是RISC组装的前提和必要条件。我们将通过层析分离纯化这个未知因子(S)，我们将其命名为RLC-X。我们将进行遗传和生化重建研究，以确切地了解RLC的形成如何促进RISC的组装。这一系列深入的研究将极大地促进我们对RNAi催化引擎holo-RISC的组成、组装和调控的理解。这些知识将极大地促进人类疾病新疗法的发展。人类疾病的起源，如癌症，通常可归因于重要基因(肿瘤抑制基因)的功能丧失和/或病理基因(癌基因)的功能获得。我们感兴趣的是了解动物细胞如何使用小RNA来沉默靶基因的表达。我们的研究不仅将促进对小RNA在生物学和疾病中的关键作用的理解，还将通过利用小RNA特异性地关闭病理基因的表达来促进人类疾病新疗法的开发。

DESCRIPTION (provided by applicant): This application addresses Broad Challenge Area (06) Enabling Technologies, Topic 06-GM-105 Small RNAs. RNA interference (RNAi) is a post-transcriptional gene silencing mechanism mediated by small interfering RNA (siRNA) or microRNA (miRNA). The significance of RNAi is underscored by its wide existence throughout metazoans and fundamental roles in biology and disease. The catalytic engine of RNAi is the RNA-induced silencing complex (RISC), which directs sequence-specific cleavage of target mRNA. A minimal RISC can form between recombinant Ago2, the ribonuclease of RISC, and single stranded siRNA, but not with double-stranded (ds) siRNA. In Drosophila, Dicer2 and R2D2 coordinately recruit nascent duplex siRNA to Ago2 to promote RISC assembly. Reconstitution of holo-RISC presents a major challenge in the RNAi field. The goal of this proposal is to understand Drosophila RISC using a biochemical fractionation and reconstitution approach. In Aim 1, we reconstituted long dsRNA-and duplex siRNA-initiated RISC activities using purified recombinant Dicer2/R2D2/Ago2 proteins. Furthermore, this core RISC reconstitution system was used to purify a novel RNAi activator that consists of Translin and Trax proteins. We will understand the mechanism by which Translin/Trax complex enhances the assembly and activity of Drosophila RISC. In Aim 2, we discovered that the RISC loading complex (RLC), whose formation precedes and is essential for RISC assembly, contained unknown factor(s) besides Dicer2/R2D2 and siRNA. We will purify this unknown factor(s), which we named as RLC-X, by chromatographic fractionation. We will perform genetic and biochemical reconstitution studies to understand exactly how the formation of RLC facilitates the assembly of RISC. This series of in-depth studies will significant advance our understanding of the composition, assembly, and regulation of holo- RISC, the catalytic engine of RNAi. The knowledge will greatly facilitate development of novel therapeutics for human diseases. The origins of human diseases, such as cancer, can be generally attributed to loss-of-function of important genes (tumor suppressor genes) and/or gain-of-function of pathological genes (oncogenes). We are interested in understanding how animal cells use small RNAs to silence target gene expression. Our studies will not only advance understanding of the critical roles of small RNAs in biology and disease, but also facilitate development of novel therapeutics for human disease by using small RNAs to specifically shut down the expression of pathological genes.