Understanding the Mechanism of RNA Interference (RNAi)

了解 RNA 干扰 (RNAi) 的机制

• 批准号: 6798458
• 负责人: PHILLIP D ZAMORE
• 金额: $ 3.98万
• 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 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6798458
• 关键词: Drosophilidae; 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）是本领域已知的技术。 双链RNA（dsRNA）在被引入细胞中时具有令人惊讶的能力， 动物来指导相应mRNA的特异性降解。在过去 两年来，RNAi已经在许多动物中被发现，包括苍蝇，蠕虫， 和小鼠，并为研究基因功能提供了新的工具， 功能基因组学越来越多的证据表明，RNAi的细胞功能是 通过抑制转座子的跳跃来维持基因组的完整性。" RNAi机制还可以用于保护细胞免受病毒感染， 甚至可能调节生殖细胞的基因表达。开始 为了了解RNAi的机制，我们最近开发了一种无细胞的 来自果蝇胚胎的系统，重现了RNAi的许多特征。 利用这种体外系统，我们已经开始了RNAi的分子分析。 在本提案中，我们的具体目标是（1）了解 dsRNA决定靶mRNA上的切割位点;（2）发现 dsRNA被加工以产生“指导RNA”的酶机制 （3）确定ATP在靶mRNA切割中的作用， RNAi途径;以及（4）鉴定组成RNAi的蛋白质 机械.这些研究有望帮助我们更好地理解这一机制， RNAi机器识别并处理dsRNA，然后使用 dsRNA中的信息以靶向特定mRNA进行破坏。我们 实验也可能有助于破译选择有效dsRNA的规则， 沉默特异性基因表达和设计dsRNA的集合， 功能基因组研究。最后，由于对RNAi的详细了解是 这是开发在哺乳动物中诱导RNAi的dsRNA类似物的先决条件， 不引起非特异性抗病毒反应，我们的工作最终可能会加速 开发基于dsRNA的人类疾病疗法。