CATALYTIC ROLES OF THE SMALL NUCLEAR RNAS IN 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， 一个由几个小的、动态的、大的复合体组成的集合体 核RNA（snRNA）和许多蛋白质。 核前mRNA剪接 通过两个协同的酯交换反应发生， 在机制上类似于II组内含子的自我剪接。 因为II组内含子在体外的自我剪接不需要 核苷酸或蛋白质辅因子，认为核前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结构。 目标 这一建议的目的是定义构象变化，更高的顺序， 参与核前体mRNA催化的RNA相互作用 拼接 核前体mRNA剪接涉及大量构象 snRNA的变化可能是由以下物质的相互作用促进的： RNA结合蛋白、RNA解旋酶或其他RNA。 早在splicesome 组装，U2 snRNA碱基巴黎与内含子分支位点序列。 随后U2和U6 RNA的碱基配对被认为是 并置这些RNA的元件和内含子底物， 催化作用 然而，已知的U2和U6 snRNA的相互作用， 前体mRNA底物不足以适当地结合和定向 分支位点腺苷残基，用于其5'端的亲核攻击 在剪接的第一步中的剪接位点。 分支部位腺苷 也至少间接参与了第二步的拼接，但 它在这一步骤中的作用人们知之甚少。 利用化学探测RNA结构的方法， 将鉴定酵母snRNAs的动态构象变化 与splicesome组装有关。 因子参与 构象变化将使用提取物进行生物化学鉴定 由阻止剪接复合体组装的突变体制备。 其他 将通过U2 RNA的遗传分析来确定相关因素。 突变体积累的复合物是前体的活性 很好 更高阶的相互作用， 定位用于催化的前mRNA分支位点腺苷将 通过相互的生化和遗传方法鉴定。 的 这些研究的结果将大大提高人们对 核前体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.