CATALYTIC ROLES OF THE SMALL NUCLEAR RNAS IN SPLICING

描述(改编自申请者摘要)：核前基因 剪接是真核基因表达中必不可少的一步，需要 一个大型的、动态的复合体的集合，由几个小的 核RNA(SnRNAs)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 在力学上类似于第二组内含子的自剪接。 因为第二组内含子在体外的自我剪接不需要 核苷酸或蛋白质辅助因子，被认为是核前mRNA 剪接很可能是剪接的催化步骤，并决定 它们在剪接体中的三维组织。目标是 这一建议的目的是以更高的顺序定义构象变化 参与核前-mRNA催化作用的RNA相互作用 拼接。 核前-mRNA剪接涉及大量的构象 小核糖核酸的变化可能由相互作用所促进 RNA结合蛋白、RNA解旋酶或其他RNA。在拼接的早期 组装，U2 SnRNA碱基为具有内含子分支位点序列的巴黎。 随后U2和U6 RNA的碱基配对被认为是 将这些RNA的元件与内含子底物并列在一起 催化作用。然而，已知的U2和U6单链RNA的相互作用，以及 前信使核糖核酸底物不足以正确结合和定向 分支部位5‘端腺苷残基的亲核攻击 在拼接的第一步中的拼接位置。分支部位腺苷 也至少间接参与了剪接的第二步，但 人们对它在这一步中扮演的角色知之甚少。 利用化学探测RNA结构的方法，拟议的研究 将确定酵母SnRNAs的动态构象变化 与拼接的装配有关。与此相关的因素 构象变化将通过使用萃取物进行生化鉴定。 从阻止剪接复合体组装的突变体中制备。其他 相关因素将通过对U2 RNA的遗传分析来确定 积累一种复合体的突变体，该复合体是活性物质的前体 很滑稽。本质上涉及的更高阶相互作用 定位前mRNA分支部位的腺苷将用于催化 通过互惠的生化和遗传方法进行鉴定。这个 这些研究的结果将极大地增进对 核前-mRNA剪接的机制和动力学。

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.