THE RIBONUCLEASES INVOLVED IN RNA INTERFERENCE

描述（由申请人提供）：当双链RNA （dsRNA）被引入细胞时，同源mRNA被降解，其基因被沉默。这种现象被称为RNA干扰（RNAi），是通过最近发现的一种生物学途径发生的。RNAi的早期步骤是将dsRNA切割成称为sirna的小片段。RNase III酶家族的成员被认为催化了这一反应，本提案的目标是了解这些酶是如何参与这一过程的。这一目标将通过表征RNase III家族成员的内在生化特性，并将这些数据与使用秀丽隐杆线虫在体内制造的RNAi观察结果相关联来实现。野生型动物和含有两种秀丽隐杆线虫RNase III基因缺失的动物之间的比较将用于体内和体外数据的关联。GFP报告结构和免疫荧光将用于确定酶的表达模式。将检测突变动物、从这些动物中提取的提取物和纯化的酶产生sirna的能力。这些酶的核酸结合特性及其RNA切割产物的结构将用于区分RNase III最近的晶体结构和现有的RNAi信息所提出的机制。缺乏RNase III酶的秀丽隐杆线虫菌株的表型将被表征，目的是为了确定酶在核途径中的作用。微阵列技术将用于寻找天然的dsRNA底物及其靶标，并在最近的筛选中鉴定出的基因将用于基因沉默的核功能测试。这些研究将为使用RNAi作为治疗剂提供有用的信息，并进一步了解这一最近发现的生物学途径。理解这种生物学途径对于理解它如何与医学相关途径交叉至关重要。

DESCRIPTION (provided by applicant): When double-stranded RNA (dsRNA) is introduced into a cell, the cognate mRNA is degraded and its gene is silenced. This phenomenon is called RNA interference (RNAi) and occurs through a recently discovered biological pathway. An early step in RNAi is cleavage of the dsRNA to small pieces called siRNAs. Members of the RNase III family of enzymes are thought to catalyze this reaction, and the goal of this proposal is to understand how these enzymes participate in the process. This goal will be approached by characterizing the intrinsic biochemical properties of RNase III family members, and correlating these data with observations about RNAi made in vivo using Caenorhabditis elegans. Comparisons between wild-type animals, and animals containing deletions in each of the two C. elegans RNase III genes, will be used to correlate in vivo and in vitro data. GFP reporter constructs and immunofluorescence will be used to determine the expression patterns of the enzymes. Mutant animals, extracts prepared from these animals, and purified enzymes, will be assayed for their ability to produce siRNAs. Nucleic acid binding properties of the enzymes, and the structure of their RNA cleavage products, will be used to distinguish between mechanisms suggested by a recent crystal structure of RNase III and existing information about RNAi. Phenotypes of C. elegans strains lacking the RNase III enzymes will be characterized with an aim towards defining the roles of the enzymes in nuclear pathways. Microarray technology will be used to search for natural dsRNA substrates and their targets, and genes identified in a recent screen will be tested for nuclear functions in gene silencing. These studies will lead to information that will be helpful in using RNAi as a therapeutic agent, and also offer further insight into this recently discovered biological pathway. Understanding this biological pathway is crucial to understanding how it intersects with medically relevant pathways.