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，需要由几个小部件组成的大型动态综合体的组装核 RNA (snRNA) 和大量蛋白质。核前mRNA剪接通过两个协同的酯交换反应发生，其方式为在机制上类似于第二组内含子的自剪接。因为 II 族内含子在体外的自剪接不需要核苷酸或蛋白质辅助因子，据认为核前 mRNA 剪接可能是剪接的催化步骤，并确定它们在剪接体内的三维组织。目标该提案的目的是定义构象变化，以及更高阶参与核前 mRNA 催化的 RNA 的相互作用拼接。核前mRNA剪接涉及大量构象 snRNA 的变化可能是由以下相互作用促进的 RNA 结合蛋白、RNA 解旋酶或其他 RNA。剪接体早期组装时，U2 snRNA 碱基与内含子分支位点序列相匹配。 U2 和 U6 RNA 的后续碱基配对被认为将这些 RNA 的元件与内含子底物并置催化。然而，U2 和 U6 snRNA 的已知相互作用，以及前 mRNA 底物不足以正确结合和定向分支位点腺苷残基用于 5' 处的亲核攻击剪接第一步中的剪接位点。分支位点腺苷也至少间接参与了拼接的第二步，但是人们对它在这一步中的作用知之甚少。使用化学探测 RNA 结构的方法，拟议的研究将识别酵母 snRNA 的动态构象变化与剪接体组装有关。涉及的因素构象变化将使用提取物进行生化鉴定由阻止剪接复合物组装的突变体制备。其他所涉及的因素将通过 U2 RNA 的遗传分析来确定积累复合物的突变体，该复合物是活性物质的前体剪接体。涉及适当的高阶相互作用定位用于催化的前 mRNA 分支位点腺苷将是通过相互的生化和遗传学方法进行鉴定。这这些研究的结果将显着增进人们的理解核前 mRNA 剪接的机制和动力学。