Biochemical Studies of Drosophila RNA-induced Silencing Complex

果蝇 RNA 诱导沉默复合物的生化研究

• 批准号: 7943057
• 负责人: QINGHUA LIU
• 金额: $ 38.3万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7943057
• 关键词: Address; Animals; Area; Biochemical; Biology; Catalytic Domain; Cells; Complex; Development; Disease; Double-Stranded RNA; Drosophila genus; Fractionation; Gene Expression; Genes; Genetic; Goals; Knowledge; Malignant Neoplasms; Mediating; Messenger RNA; MicroRNAs; Molecular; Names; Oncogenes; Proteins; RNA Interference; RNA-Induced Silencing Complex; Recombinants; Recruitment Activity; Regulation; Research; Research Proposals; Ribonucleases; Role; Series; Small Interfering RNA; Small RNA; System; Technology; Tumor Suppressor Genes; base; gain of function; human DICER1 protein; human disease; in vivo; insight; interest; loss of function; novel; novel therapeutics; reconstitution; success

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.

描述（由申请人提供）： 该申请涉及广泛的挑战领域（06）促成技术，主题06 gm-105小型RNA。 RNA干扰（RNAi）是一种通过小型干扰RNA（siRNA）或microRNA（miRNA）介导的转录后基因沉默机制。 RNAi的意义因其在生物学和疾病中的基本角色而突显了其广泛的存在。 RNAi的催化发动机是RNA诱导的沉默复合物（RISC），它指导靶mRNA的序列特异性裂解。在重组AGO2，RISC的核糖核酸酶和单链siRNA之间可以形成最小的RISC，但不能与双链（DS）siRNA形成。在果蝇中，DICER2和R2D2协同招募新生的双工siRNA至AGO2，以促进RISC集会。 Holo-Risc的重建提出了RNAi领域的重大挑战。该提案的目的是使用生化分馏和重建方法来了解果蝇RISC。在AIM 1中，我们使用纯化的重组DICER2/R2D2/AGO2蛋白来重构长dsRNA和双链siRNA引起的RISC活性。此外，该核心RISC重构系统用于净化由Translin和Trax蛋白组成的新型RNAi激活剂。我们将了解转化/TRAX复合物增强果蝇RISC的组装和活性的机制。在AIM 2中，我们发现RISC载荷复合物（RLC）的形成在RISC组件之前是必不可少的，除了DICER2/R2D2和siRNA外，还包含未知因子。我们将通过色谱分离净化这个未知因子，将其称为RLC-X。我们将进行遗传和生化重构研究，以准确了解RLC的形成如何促进RISC的组装。这一系列深度研究将有很大的重大理解RNAi的催化发动机的全体构成和调节的理解。这些知识将极大地促进针对人类疾病的新型治疗学的发展。人类疾病（例如癌症）的起源通常归因于重要基因（肿瘤抑制基因）和/或病理基因（Oncogenes）功能的功能丧失。我们有兴趣了解动物细胞如何使用小的RNA沉默靶基因表达。我们的研究不仅会提高小RNA在生物学和疾病中的关键作用，而且还可以通过使用小RNA专门关闭病理基因的表达来促进针对人类疾病的新型治疗。