Understanding the Mechanism of RNA Interference (RNAi)

描述(申请人描述)：RNA干扰(RNAi)是 双链RNA(DsRNA)被引入到 动物可以直接特定降解相应的mRNA。在最后一次 两年来，已经在许多动物中发现了RNAi，包括苍蝇、蠕虫、 和小鼠，为研究基因功能和 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“跳跃”来维持基因组的完整性。 RNAi机制还可能用于保护细胞免受病毒感染和 甚至可能是为了调节生殖系中的基因表达。开始，开始 了解RNAi的机制，我们最近开发了一种无细胞 来自果蝇胚胎的系统，概括了RNAi的许多特征。 利用这个体外系统，我们已经开始了对RNAi的分子分析。 在这项建议中，我们的具体目标是(1)了解 DsRNA决定了靶mrna上的切割位点；(2)发现 DsRNA被加工成“引导RNA”的酶机制 直接切割靶基因；(3)确定三磷酸腺苷在 RNAi途径；以及(4)鉴定组成RNAi的蛋白质 机械设备。这些研究承诺通过以下方式帮助我们更好地理解这种机制 RNAi机制识别和处理dsRNA，然后使用 DsRNA中的信息以特定的mRNA为靶点进行破坏。我们的 实验也可能有助于破译选择有效dsRNA的规则 沉默特定的基因表达和设计dsRNA集合 功能基因组学研究。最后，由于对RNAi的详细理解是 开发在哺乳动物中诱导RNAi的dsRNA类似物的先决条件 不会引起非特异性的抗病毒反应，我们的工作最终可能会加快 基于dsRNA的人类疾病治疗方法的发展。

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.