Modular Structure and Function of RNase lll, a dsRNA-Binding Protein

RNase III（一种 dsRNA 结合蛋白）的模块化结构和功能

• 批准号: 9905406
• 负责人: Robert Simons
• 金额: $ 23.4万
• 依托单位: University of California-Los Angeles
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9905406&HistoricalAwards=false
• 关键词: Modular; Structure; Function; RNase; lll

SimonsRNA-binding proteins are involved in all aspects of gene expression, including replication, transcription, translation, ribosomal and messenger RNA processing and decay, RNA splicing and editing, and RNA transport and export. Ribonucleases are key players in these events. Ribonuclease III (RNase III) is a double-stranded RNA (dsRNA)-specific endonuclease that is widely conserved, playing vital or essential roles in RNA processing and decay. Each RNase III monomer (of the functioning dimer) contains a single copy of the dsRNA-binding motif (dsRBM) found in a large number of diverse proteins known or thought to bind dsRNA. RNase III also contains a highly conserved catalytic domain (catD), so far unique to this enzyme. Recent and important evidence reveals how the dsRBM binds to dsRNA as an independently folded protein module. Other recent evidence shows that catD is also an independent module, possessing robust and specific endoribonuclease activity under specific conditions. These studies suggest that the dsRBM and catD subdomains of RNase III likely interact with one another through a network of intimate contacts, to stabilize binding and to create a more efficient catalytic site. Subdomain contacts within otherwise modular proteins, including other RNA-binding proteins, are not well-understood. This research project comprises an integrated genetic assault on RNase III structure and function at the subdomain level, through well-defined specific aims. Key biochemical work will directly test genetic predictions. The principal determinants of RNase III dimerization will be examined in vivo, through the isolation of point mutations in a novel genetic screen. The potential contribution of dsRNA substrate binding to dimer stability will be carefully assessed, aided by existing well-characterized dsRBM point mutations that prevent binding. Substrate-binding determinants in catD will also be sought. Potential interactions between dsRBM and catD will be studied carefully, with a newly developed in vivo assay in which RNase III activity is reconstituted from the individually expressed subdomains, as well as with the recently developed prokaryotic two-hybrid system and other means. These efforts will yield important insights into dsRNA-binding, RNase III catalysis, and modular protein structure and function in general.SimonsRNA is one of the storehouses of genetic material in all cells. RNA-binding proteins are involved in all aspects of RNA function. RNase III is one such RNA-binding protein. It is found in most cells. RNase III contains an RNA-binding module (RBM) found in a large number of related proteins that bind RNA. RNase III also contains a catalytic module (catD), unique to this protein family. Recent and important evidence suggests that the RBM and catD modules interact with one another through a network of intimate contacts. In general, subdomain contacts in modular proteins are not well-understood. This research project will mount an integrated genetic and biochemical assault on RNase III structure and function, at the modular level. These efforts will yield important insights into RNA-binding, RNase III function, and modular protein structure and function in general.

simonsrna结合蛋白参与基因表达的各个方面，包括复制、转录、翻译、核糖体和信使RNA加工和衰变、RNA剪接和编辑、RNA转运和输出。核糖核酸酶在这些事件中起着关键作用。核糖核酸酶III （ribonucase III, RNase III）是一种广泛保守的双链RNA （dsRNA）特异性内切酶，在RNA加工和衰变过程中起着至关重要的作用。每个RNase III单体（功能二聚体）包含一个dsRNA结合基元（dsRBM）的单个拷贝，该基元存在于大量已知或被认为结合dsRNA的不同蛋白质中。RNase III还包含一个高度保守的催化结构域（catD），这是迄今为止该酶所特有的。最近的重要证据揭示了dsRBM如何作为一个独立折叠的蛋白质模块与dsRNA结合。最近的其他证据表明，catD也是一个独立的模块，在特定条件下具有强大的特异性核糖核酸内酶活性。这些研究表明，RNase III的dsRBM和catD亚结构域可能通过密切接触网络相互作用，以稳定结合并创造更有效的催化位点。在其他模块化蛋白质（包括其他rna结合蛋白）内的亚结构域接触尚未得到很好的理解。本研究项目通过明确的具体目标，在子结构域水平上对RNase III的结构和功能进行综合遗传攻击。关键的生化工作将直接检验基因预测。RNase III二聚化的主要决定因素将在体内进行检查，通过在一种新的遗传筛选中分离点突变。dsRNA底物结合对二聚体稳定性的潜在贡献将被仔细评估，通过现有的良好表征的dsRBM点突变来阻止结合。还将寻找catD中的底物结合决定因子。将仔细研究dsRBM和catD之间的潜在相互作用，通过新开发的体内试验，从单独表达的子结构域重构RNase III活性，以及最近开发的原核双杂交系统和其他方法。这些努力将对dsrna结合、RNase III催化和模块化蛋白质结构和功能产生重要的见解。SimonsRNA是所有细胞中遗传物质的储存库之一。RNA结合蛋白参与RNA功能的各个方面。RNase III就是这样一种rna结合蛋白。它存在于大多数细胞中。RNase III含有一个RNA结合模块（RBM），存在于大量结合RNA的相关蛋白中。RNase III还含有一个催化模块（catD），这是该蛋白家族所特有的。最近的重要证据表明，RBM和catD模块通过亲密接触网络相互作用。一般来说，模块化蛋白中的子结构域接触还没有被很好地理解。该研究项目将在模块化水平上对RNase III的结构和功能进行综合遗传和生化攻击。这些努力将对rna结合、RNase III功能以及模块化蛋白质结构和功能产生重要的见解。