FUNCTION OF SNRNP PARTICLES IN PREMRNA SPLICING

描述这是一份修订后的研究交互更新申请 在U1 snRNP和拼接机器的其他组件之间。 的 主要关注的是U1相关蛋白Prp 39，由 研究人员在早期筛选新的TS剪接突变体。 基于 根据观察，这种蛋白质1）是形成 承诺复合物，2）但仅与U1 snRNP弱相关， 和3）与未剪接的前体而不是中间体缔合， 研究者将检验一个中心假设，即U1 snRNP募集 随后从剪接体释放是由 弱结合蛋白的结合和解离。一个主要目标是 为了确定Prp 39和其他蛋白质在循环中的精确时间， 已知U1-snRNP蛋白（Snp 1（70 k），mud 1，mud 2）结合和解离 从U1和剪接体;这种方法依赖于 抗体和prp突变集合的可用性被阻断 在这个过程中的特定步骤。交联实验将解决 Prp 39稳定U1与5'剪接位点结合的假设 通过与前mRNA的直接相互作用，并确定在什么时候， U1/5' SS相互作用被U6/5' SS相互作用取代。 Prp 39和其他U1 snRNP蛋白之间的相互作用将被寻找 通过2-hybrid方法。Prp 39所必需的U1序列 通过诱变鉴定相互作用。一种哺乳动物同源物 将寻求免疫和遗传手段;这将允许 在较高的细胞中，它是一个非snRNP剪接因子的预测的检验 有机体Prp 39在U1 snRNA 3'端成熟中的作用 将被分析。最后，描述了一种遗传筛选， 鉴定增强剪接位点选择的基因。

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.