Reactivity epitopes of ribonuclease III substrates

描述(申请人提供)：双链RNA的酶切割是各种真核和原核RNA成熟和降解的关键步骤，也是RNA干扰(RNAi)和相关的基因沉默和基因组维护机制中的关键事件。DsRNA的切割是由核糖核酸酶III(RNase III)家族的成员催化的，这些内切核酸酶在细菌和真核细胞中高度保守。真核RNaseIII的同源基因包括DICER和DROSHA，它们实现了microRNAs和其他基因调控RNA的成熟。RNaseIII也是锥虫线粒体中RNA编辑小体的一个组成部分。细菌核糖核酸酶III加工dsRNA调节细胞和噬菌体基因的表达、质粒复制、抗生素生产和毒力因子的表达。细菌的核糖核酸酶底物以高度特异的方式被切割，这是RNA适当的半衰期或最佳功能所必需的。也有证据表明，细菌RNaseIII可以通过非催化方式与RNA结合来调节基因的表达。该项目的长期目标是表征在RNaseIII底物识别和切割中具有特异性的RNA序列和结构元件以及RNA-蛋白质相互作用。特定目标1将采用体外选择、核苷酸类似干扰作图和定点突变来鉴定识别所必需的底物功能团。特定目标2将表征蛋白质官能团对底物专一性和结合能的贡献。定点突变和多肽片段交换实验将确定与结合特异性有关的RNaseIII结构域。具体目标3将确定两个特定RNA中催化反式决定簇的序列和结构特征。在体外选择、定点突变和RNA结构探测将被用来确定体外选择的、具有结合能力的RNA和波长噬菌体CIII mRNA5‘-前导的关键结构和功能特征。核糖核酸酶III的激活机制将使用体外翻译系统进行分析。除了大肠杆菌RNase III，该项目还将使用Aquifex aeolicus RNase III和Thermotoga maritima RNase III，这些RNase III的结构数据是可用的。这些分析将有助于确定RNaseIII在dsRNA处理中如何实现特异性，并最终确定dsRNA处理在细胞防御机制中的作用，包括RNAi和特定疾病状态，包括癌症和传染病。

DESCRIPTION (provided by applicant): The enzymatic cleavage of double-stranded RNA is an essential step in the maturation and degradation of diverse eukaryotic and prokaryotic RNAs, and is a key event in RNA interference (RNAi) and related gene silencing and genome maintenance mechanisms. dsRNA cleavage is catalyzed by members of the ribonuclease III (RNase III) family of endoribonucleases, which are highly conserved in bacterial and eukaryotic cells. Eukaryotic RNase III orthologs include Dicer and Drosha, which carry out the maturation of microRNAs and other gene-regulatory RNAs. RNase III also is a component of RNA editosomes in trypanosome mitochondria. dsRNA processing by bacterial ribonucleases III regulates cellular and phage gene expression, plasmid replication, antibiotic production, and virulence factor expression. Bacterial RNase substrates are cleaved in a highly site-specific manner, which is required for proper half-life or optimal function of the RNAs. There also is evidence that bacterial RNase III can regulate gene expression by binding RNA in a non-catalytic mode. The long-term objective of this project is to characterize the RNA sequence and structural elements and RNA-protein interactions that confer specificity in RNase III substrate recognition and cleavage. Specific Aim 1 will employ in vitro selection, nucleotide analog interference mapping and site-directed mutagenesis to identify substrate functional groups essential for recognition. Specific Aim 2 will characterize protein functional group contributions to substrate specificity and binding energy. Site-directed mutagenesis and polypeptide segment exchange experiments will identify RNase III domains involved in specificity of binding. Specific Aim 3 will define the sequence and structural features of catalytic antideterminants in two specific RNAs. In vitro selection, site-directed mutagenesis, and RNA structure-probing will be used to determine the critical structural and functional features of an in vitro-selected, binding-competent RNA, and the lambda phage cIII mRNA 5'-leader. The mechanism of RNase III activation will be analyzed using an in vitro translation system. Along with Escherichia coli RNase III, this project will employ Aquifex aeolicus RNase III and Thermotoga maritima RNase III, for which structural data is available. These analyses will serve to define how specificity is achieved in dsRNA processing by RNase III, and ultimately determine the role of dsRNA processing in the cellular defense mechanisms, including RNAi and in specific disease states, including cancer and infectious diseases.