CATALYTIC ROLES OF THE SMALL NUCLEAR RNAS IN SPLICING

小核 RNA 在剪接中的催化作用

基本信息

批准号：
2701648
负责人：
David Scott McPheeters
金额：
$ 17.84万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-05-01 至 1999-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2701648
关键词：
RNA splicing catalyst conformation fungal genetics mutant nucleic acid structure precursor mRNA small nuclear RNA small nuclear ribonucleoproteins spliceosomes suppressor mutations

项目摘要

DESCRIPTION (Adapted from Applicant's Abstract): Nuclear pre-mRNA splicing is an essential step in eukaryotic gene expression and requires the assembly of a large, dynamic complex consisting of several small nuclear RNAs (snRNAs) and numerous proteins. Nuclear pre-mRNA splicing occurs via two concerted transesterification reaction in a manner that is mechanistically similar to the self-splicing of group II intron. Because the self-splicing of group II introns in vitro requires no nucleotide or protein cofactors, it is thought that nuclear pre-mRNA splicing is likely to be catalytic steps of splicing, and to determine their three-dimensional organization within the spliceosome. The goal of this proposal is to define conformational changes in, and higher order interaction of, the RNAs involved in catalysis of nuclear pre-mRNA splicing. Nuclear pre-mRNA splicing involves a large number of conformational changes in the snRNAs that may be facilitated by the interactions of RNAs binding proteins, RNA helicase, or other RNAs. Early in splicesome assembly, the U2 snRNA base paris with the intron branch site sequence. Subsequent base pairing of the U2 and U6 RNAs is then thought to juxtapose elements of these RNAs and the introns substrate for catalysis. However, the known interactions of the U2 and U6 snRNAs, and the pre-mRNA substrate are insufficient to properly bind and orient the branch site adenosine residue for its nucleophilic attack at the 5' splice site during the first step of splicing. The branch site adenosine is also at least indirectly involved in the second step of splicing, but its role in this step is poorly understood. Using methods for chemically probing RNA structure, the proposed research will identify dynamic conformational changes in the yeast snRNAs associated with splicesome assembly. Factors involved in the conformational changes will be identified biochemically using extracts prepared from mutants which arrest splicing complex assembly. Other factors involved will be identified by genetic analysis of U2 RNA mutants that accumulate a complex which is the precursor to the active splicesome. Higher order interactions involved in the proper positioning the pre-mRNA branch site adenosine for catalysis will be identified through reciprocal biochemical and genetic approaches. The results of these studies will significantly increase the understanding of the mechanism and dynamics of nuclear pre-mRNA splicing.

描述（改编自申请人的摘要）：核前MRNA 剪接是真核基因表达的重要步骤，需要大型动态复合物的组装，由几个小核RNA（SNRNA）和许多蛋白质。核前mRNA剪接通过两个协同的式式式反应，以某种方式发生在机械上类似于II组内含子的自剪接。因为体外II组内含子的自拼图不需要核苷酸或蛋白质辅因子，据认为核前mRNA 剪接可能是拼接的催化步骤，并确定他们在剪接体中的三维组织。目标该提议的内容是定义构象变化和更高阶段与核前mRNA催化的RNA相互作用剪接。核前mRNA剪接涉及大量构象 snRNA的变化可能会通过 RNA结合蛋白，RNA解旋酶或其他RNA。早期组件，带有内含子分支位点序列的U2 SnRNA基础巴黎。然后认为U2和U6 RNA的随后基础配对这些RNA的并置元素和内含子基板催化。但是，U2和U6 SNRNA的已知相互作用，以及前MRNA底物不足以适当结合和定向分支位点腺苷残基，用于5' 在剪接的第一步中拼接站点。分支部位腺苷也至少间接参与了拼接的第二步，但是它在这一步骤中的作用知之甚少。使用化学探测RNA结构的方法，拟议的研究将确定酵母SNRNA的动态构象变化与拼接组件相关联。涉及的因素构象变化将使用提取物进行生化鉴定由停止剪接复合物组件的突变体制备。其他涉及的因素将通过对U2 RNA的遗传分析来确定积累复合物的突变体是活性的前体拼接。适当涉及的高阶互动将催化前的mRNA分支部位腺苷定位为通过相互生化和遗传方法鉴定。这这些研究的结果将大大增加理解核前mRNA剪接的机理和动力学。