Ribonuclease III mechanism of double-strand RNA cleavage

描述(申请人提供)：双链RNA的酶裂解是许多真核和原核RNA成熟和腐烂的关键步骤。双链(DS)RNA裂解是由核糖核酸酶I11超家族的成员进行的。RNaseIII同源基因参与了mRNA和rRNA的成熟、mRNA的降解和反义RNA的作用。最近的一个重要发现是RNaseIII同源基因“Disher”在RNA干扰(RNAi)中的中心参与。RNAi被认为可以抑制病毒感染，抑制逆转录病毒的运动，并参与包括人类在内的真核生物的特定发育途径。高度保守的序列和结构元素的出现表明RNase Ill同源物使用相同的催化机制，这一点尚不清楚。核糖核酸酶III超家族中研究最多的成员是大肠杆菌核糖核酸酶III，最近在确定大肠杆菌核糖核酸酶III的作用机制方面取得了重大进展。本项目的长期目标是确定大肠杆菌RNase Ill对dsRNA的切割机制。该项目的具体目标是：(1)确定保守氨基酸在催化结构域中的功能作用。为了做到这一点，将在底物结合和切割试验中测试具有保守催化结构域残基替换的RNase Ill突变体；(2)确定二价金属离子的化学计量比和在催化中的功能。为此，将使用RNase Ill和选定的突变体进行裂解动力学分析和金属离子结合分析；(3)确定RNase Ill双链RNA结合域(DsRBD)中提供结合能和优化催化效率的序列和结构特征。这将通过分析特定dsRBD突变体的RNA结合和噬菌体展示来完成；(4)确定RNase Ill底物复合体的化学计量比，并确定对RNase Ill功能重要的亚基间相互作用。将使用人工杂二聚体进行凝胶过滤、离心法、底物结合和裂解试验。这些目标的实现不仅将提供有关E.coil RNase Ill的重要信息，还将从机制上深入了解RNase Ill在真核RNA加工和降解途径(包括RNAi)中的同源功能。

DESCRIPTION (provided by applicant): The enzymatic cleavage of double-stranded RNA is an essential step in the maturation and decay of many eukaryotic and prokaryotic RNAs. Double-stranded (ds) RNA cleavage is carried out by members of the ribonuclease Ill superfamily of endoribonucleases. RNase III orthologues participate in mRNA and rRNA maturation, mRNA degradation, and antisense RNA action. An important recent finding is the central involvement of the RNase III orthologue "Dicer" in RNA interference (RNAi). RNAi is proposed to suppress viral infection, inhibit retroposon movement, and participate in specific developmental pathways in eukaryotes, including humans. The occurrence of highly conserved sequences and structural elements indicate that RNase Ill orthologues use the same catalytic mechanism, which is unknown. The most studied member of the RNase III superfamily is Escherichia coli ribonuclease III and there has been recent significant progress in determining the E. coil RNase Ill mechanism of action. The long-range goal of this project is to determine the mechanism of dsRNA cleavage by E. coil RNase Ill. The specific aims of the project are to: (1) determine the functional roles of conserved amino acids in the catalytic domain. To accomplish this, RNase Ill mutants with substitutions of conserved catalytic domain residues will be tested in substrate binding and cleavage assays; (2), determine divalent metal ion stoichiometry and function in catalysis. To accomplish this, kinetic assays of cleavage, and metal ion binding assays using RNase Ill and selected mutants will be performed; (3), identify sequence and structural features in the RNase Ill double-stranded-RNA-binding domain (dsRBD) that confer binding energy and optimize catalytic efficiency. This will be accomplished by analyzing RNA binding of specific dsRBD mutants, and by phage display; (4), determine the stoichiometry of an RNase Ill-substrate complex, and identify intersubunit interactions important for RNase Ill function. Gel filtration, centrifugation, and substrate binding and cleavage assays using artificial heterodimers will be performed. Accomplishing these aims not only will provide important information on E. coil RNase Ill, but will also provide mechanistic insight on RNase Ill orthologue function in eukaryotic RNA processing and degradation pathways, including RNAi.