Understanding the Mechanism of RNA Interference (RNAi)

了解 RNA 干扰 (RNAi) 的机制

• 批准号: 6871960
• 负责人: PHILLIP D ZAMORE
• 金额: $ 24.35万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2006-07-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6871960
• 关键词: Drosophilidae; RNA interference; SDS polyacrylamide gel electrophoresis; adenosine triphosphate; affinity chromatography; binding sites; cell free system; chemical cleavage; double stranded RNA; enzyme mechanism; gel filtration chromatography; gene expression; gene induction /repression; genetic mapping; green fluorescent proteins; messenger RNA; molecular cloning; molecular dynamics; nucleic acid metabolism; polymerase chain reaction; radiotracer; ribonuclease III; sedimentation velocity

DESCRIPTION (Applicant's Description): RNA interference (RNAi) is the surprising ability of double-stranded RNA (dsRNA) when introduced into an animal to direct the specific degradation of a corresponding mRNA. In the last two years, RNAi has been identified in many animals, including flies, worms, and mice, and has provided a new tool for studying gene function and for functional genomics. Evidence is mounting that the cellular function of RNAi is to maintain the integrity of the genome by suppressing transposon "jumping." The RNAi machinery may also serve to defend cells against viral infection and perhaps even to regulate gene expression in the germ line. To begin to understand the mechanism underlying RNAi, we recently developed a cell-free system from Drosophila embryos that recapitulates many of the features of RNAi. Using this in vitro system, we have begun a molecular analysis of RNAi. In this proposal, our specific goals are (1) to understand how the sequence of the dsRNA determines the sites of cleavage on the target mRNA; (2) to discover the enzymatic mechanism by which the dsRNA is processed to create "guide RNAs" that direct cleavage of the target mRNA; (3) to determine the role of ATP in the RNAi pathway; and (4) to identify the proteins that compose the RNAi machinery. These studies promise to help us better understand the mechanism by which the RNAi machinery recognizes and processes dsRNA and then uses the information in the dsRNA to target a specific mRNA for destruction. Our experiments may also help to decipher the rules for selecting potent dsRNAs for silencing specific gene expression and in designing collections of dsRNAs for functional genomic studies. Finally, since a detailed understanding of RNAi is a prerequisite for developing dsRNA analogs that induce RNAi in mammals but do not provoke non-specific anti-viral responses, our work may ultimately speed the development of dsRNA-based therapies for human diseases.

描述（申请人的描述）：RNA干扰（RNAi）是 当引入双链RNA（DSRNA）的惊人能力 动物指导相应mRNA的特定降解。在最后 两年来，RNAi在许多动物中都被发现，包括苍蝇，蠕虫， 和小鼠，并为研究基因功能和用于研究的新工具 功能基因组学。有证据表明RNAi的细胞功能是 通过抑制转座子“跳跃”来维持基因组的完整性。 RNAi机械还可以用于防御病毒感染，并 甚至可能调节种系中的基因表达。开始 了解RNAi的机制，我们最近开发了一个无细胞 果蝇胚胎的系统概括了RNAi的许多特征。 使用此体外系统，我们已经开始对RNAi进行分子分析。 在此提案中，我们的具体目标是（1）了解 dsRNA确定靶mRNA上裂解的位点； （2）发现 处理DSRNA以创建“指导RNA”的酶促机制 靶mRNA的直接切割； （3）确定ATP的作用 RNAi途径； （4）鉴定构成RNAi的蛋白质 机械。这些研究有望帮助我们更好地了解 RNAi机械识别和处理DSRNA，然后使用 DSRNA中的信息以靶向特定的mRNA进行破坏。我们的 实验也可能有助于解读选择有效DSRNA的规则 沉默特定基因表达和设计DSRNA的集合 功能性基因组研究。最后，由于对RNAi的详细理解是 开发DSRNA类似物的先决条件 不引起非特异性的反病毒反应，我们的工作最终可能会加快 基于DSRNA的人类疾病疗法的发展。