FUNCTION OF SNRNP PARTICLES IN PREMRNA SPLICING

说明：这是研究交互作用的修订续订应用程序 U1 SnRNP与拼接机械的其他部件之间的连接。这个 主要关注的是一种与U1相关的蛋白质Prp39，由 研究人员在早期筛选出新的ts剪接突变体。基座 根据观察，这种蛋白质1)是形成 承诺复合体，2)但仅与U1 SnRNPs弱相关， 3)与未剪接体前体结合，而不与中间体结合 研究人员将检验一项中心假设，即U1 SnRNP招募到 随后从剪接体的释放是由 弱结合蛋白的结合和解离。一个主要目标是 以确定Prp39和其他元素在周期中的准确时间 已知的U1-SnRNP蛋白(Snp1(70k)、MUD1、MUD2)结合和解离 来自U1和剪接体；此方法依赖于 抗体的可用性和一组被阻止的PRP突变 在这个过程中的特定步骤。交联性实验将解决 Prp39稳定U1与5‘剪接位点结合的假说 通过直接与前信使核糖核酸相互作用，确定在什么点上 U1/5‘SS互作被U6/5’SS互作所取代。 Prp39与其他U1 SnRNP蛋白之间的相互作用将被研究 通过2-混合方法。Prp39必需的U1序列 相互作用将通过突变来确定。一种哺乳动物的同源物 将通过免疫和遗传手段寻求；这将允许 预测它是一个非SNRNP剪接因子在较高的 有机体。Prp39在U1单链RNA 3‘端成熟中的作用 将会被分析。最后，描述了一个基因筛查，以允许 增强剪接位点选择的基因的鉴定。

DESCRIPTION This is a revised renewal application to study interactions between U1 snRNP and other components of the splicing machinery. The major focus is on a U1-associated protein, Prp39, identified by the investigator in an earlier screen for novel ts splicing mutants. Based on the observations that this protein 1) is required for formation of commitment complexes, 2) yet is only weakly associated with U1 snRNPs, and 3) associates with unspliced precursor but not intermediates, the investigator will test a central hypothesis that U1 snRNP recruitment to and subsequent release from the spliceosome is mediated by the association and dissociation of weakly bound proteins. A major goal is to determine the precise times in the cycle at which Prp39 and other known U1-snRNP proteins (Snp1 (70k), mud1, mud2) associate and dissociate from U1 and from the spliceosome; this approach relies on the availability of antibodies and of a collection of prp mutations blocked at specific steps in the pathway. Crosslinking experiments will address the hypothesis that Prp39 stabilizes U1 binding to the 5' splice site by direct interaction with the pre-mRNA and determine at what point the U1/5' SS interaction is replaced by the U6/5'SS interaction. Interactions between Prp39 and other U1 snRNP proteins will be sought by the 2-hybrid approach. Sequences in U1 necessary for Prp39 interaction will be identified by mutagenesis. A mammalian homologue will be sought by immunological and genetic means; this will allow a test of the prediction that it is a non-snRNP splicing factor in higher organisms. The role of Prp39 in maturation of the 3' end of U1 snRNA will be analyzed. Finally, a genetic screen is described to allow identification of genes that enhance splice site selection.