Regulatory Mechanisms of RNA Interferance

RNA干扰的调控机制

• 批准号: 8050572
• 负责人: QINGHUA LIU
• 金额: $ 27.7万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8050572
• 关键词: Address; Binding Proteins; Biochemical; Biological; Biology; Data; Disease; Double-Stranded RNA; Drosophila genus; Fractionation; Gene Silencing; Genetic; Genetic Screening; Genome; Goals; Human; Knowledge; Mediating; Messenger RNA; MicroRNAs; Mitogen-Activated Protein Kinases; Molecular; Pathway interactions; Phosphorylation; Positioning Attribute; Post-Translational Regulation; Process; Proteins; RNA; RNA Interference; RNA Interference Pathway; RNA-Induced Silencing Complex; Recombinants; Regulation; Research; Research Proposals; Role; Science; Small Interfering RNA; System; Technology; Testing; Translational Repression; base; design; fly; human DICER1 protein; human disease; improved; innovation; insight; mutant; novel; novel therapeutics; reconstitution

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.

描述（由申请人提供）：该研究建议的总体目标是了解RNA干扰的调节机制（RNAI）。 RNAi是通过microRNA（miRNA）和小干扰RNA（siRNA）介导的转录后基因分解机制。尽管已经确定了许多RNAi组件，但它们在RNAi中的特定功能尚不清楚。迄今为止，只有三个核心组件经过生化验证。 DICER在siRNA和miRNA上进行长dsRNA和前miRNA。在siRNA或miRNA的指导下，Argonaute（AGO）指导序列特异性的裂解和翻译抑制同源mRNA。 dsRNA结合蛋白（DSRBP），例如蝇中的R2D2，促进了siRNA从dicer到AGO的转移，以形成效应RNA诱导的沉默（RISC）复合物。 生化分馏和重构是一种理解RNAi深入分子机制的强大而必不可少的方法。在当前的研究中，我们建议通过使用重组DICER/DSRBP/AGO蛋白来重建Holo-Risc活动。我们的重建制度将使我们处于独特的立场，以解决该领域的几个基本和长期存在的问题：哪些因素构成了Holo-Risc？ Risc如何组装？ RNAi如何调节？为了实现我们的目标，我们设计了三个特定的研究途径。在AIM 1中，我们使用纯化的重组DCR-2/R2D2/AGO2蛋白来重构果蝇dsRNA引起的RISC活性。这种重组制度将使我们能够研究RISC议会的深入生化机制。在AIM 2中，我们将采用一种组合性的遗传和生化方法来鉴定果蝇RNAi的新调节剂，并研究RNAi调节的生化机制。在AIM 3中，我们将使用重组DICER/DSRBP/AGO2蛋白重新构建人类全率活性，并通过DSRBP的磷酸化研究人RNAi的调节。 RNAi及相关途径是控制基因组活性的基本和全球机制。 RNAI调节机制的研究将把田地连接到更广泛的生物环境，并为生物学和疾病中小调节性RNA的正常和病理法规提供生化基础。另一方面，对RNAi的机械理解将促进新的和改进的基因沉默技术的合理设计。 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，从了解基本生物学，揭示人类疾病的分子基础到发展新型疗法。

项目成果

Expression, purification, and analysis of recombinant Drosophila Dicer-1 and Dicer-2 enzymes.

• DOI: 10.1007/978-1-59745-191-8_2
• 发表时间: 2008
• 期刊: Methods in molecular biology
• 作者: Xuecheng Ye;Qinghua Liu

Structure of C3PO and mechanism of human RISC activation.

• DOI: 10.1038/nsmb.2032
• 发表时间: 2011-06
• 期刊: Nature structural & molecular biology
• 影响因子: 16.8

ATM signals miRNA biogenesis through KSRP.

ATM 通过 KSRP 向 miRNA 生物发生发出信号。

• DOI: 10.1016/j.molcel.2011.01.027
• 发表时间: 2011
• 期刊: Molecular cell
• 影响因子: 16
• 作者: Liu,Ying;Liu,Qinghua
• 通讯作者: Liu,Qinghua