In Vivo Analysis of Spliceosomal Protein Function

描述(由申请人提供)：拟议研究的目标是 描述参与剪接的蛋白质在体内的功能 监管。使用性别特定的剪接调控的例子 果蝇二元开关基因SXL的研究表明， Sans-fille(SnF)，人类U1nRNP-U1a和U2nRNP-U2b的对应物 蛋白质在SXL剪接自我调节中起着关键作用。虽然很明显 SNF和女性特有的SXL蛋白物理上相互作用并被发现 在同一个多组分的复合体中，这个成员的身份 复合体以及它们如何组装以抑制男性特有的剪接 外显子还有待确定。 为了解决这些问题，提出了三个具体目标。在目标1中， 假说，基于遗传和蛋白质相互作用研究的数据， SPP-87B和SIN在SXL剪接自动调节中发挥不可或缺的作用将是 通过一种包括蛋白质定位研究的综合方法进行测试， 蛋白质-蛋白质相互作用研究和功能丧失的遗传分析 突变。在目标2中，在SXL剪接中起作用的其他蛋白质 自动调节将通过利用研究中的信息来确定 酵母和哺乳动物细胞在同源剪接因子中产生突变 并测试它们的遗传交互作用和未能持续跳过 SXL男性外显子。尚未连接到剪接的候选基因座 将在全基因组遗传筛查和生化筛查中确定 核实。在目标3中，SNF及其合作伙伴的生化特性 对女性特异性SXL剪接重要的蛋白质将使用 蛋白质-蛋白质和蛋白质-RNA相互作用分析。此外，分子 控制雄性外显子利用的途径将通过识别 哪些交互依赖于SXL和/或关联的存在 在SXL和SNF之间使用突变动物的提取物。 来自这三个目标的组合数据将为SXL如何、 SNF和他们的蛋白质伙伴组装成阻断复合体，并 共同作用促进男性外显子跳跃。更重要的是，因为 果蝇和果蝇之间已知剪接因子的显著保守性 人类，从这些研究中获得的信息将显著进步 我们对脊椎动物受调控剪接的理解。

DESCRIPTION (provided by applicant): The goal of the proposed research is to delineate the functions in vivo of proteins that participate in splicing regulation. Using the example of sex-specific splicing regulation of the Drosophila binary switch gene Sex-lethal (Sxl), studies have shown that SANS-FILLE (SNF), the counterpart of the human U1 snRNP-U1A and U2 snRNP-U2B" proteins, plays a key role in Sxl splicing autoregulation. While it is clear that SNF and the female-specific SXL protein physically interact and are found in the same multi-component complex, the identity of the other members of this complex and how they are assembled to inhibit splicing of the male-specific exon remains to be determined. To address these issues, three specific aims are proposed. In Aim 1, the hypothesis, based on data from genetic and protein-protein interaction studies, that SPP-87B and SIN play integral roles in Sxl splicing autoregulation will be tested by a combined approach that will include protein localization studies, protein-protein interaction studies, and genetic analysis of loss-of-function mutations. In Aim 2, additional proteins that function in Sxl splicing autoregulation will be identified by exploiting information from studies in yeast and mammalian cells to create mutations in orthologous splicing factors and testing them for both genetic interactions and failure to consistently skip the Sxl male-exon. Candidate loci that have not yet been connected to splicing will be identified in a genome-wide genetic screen coupled with biochemical verification. In Aim 3, the biochemical properties of SNF and its partner proteins important for female-specific Sxl splicing will be identified using protein-protein and protein-RNA interaction assays. In addition, the molecular pathway that controls male-exon utilization will be elucidated by identifying which interactions are dependent on the presence of SXL and/or the association between SXL and SNF using extracts from mutant animals. The combined data from these three aims will provide new insight into how SXL, SNF and their protein partners are assembled into a blocking complex and function together to promote male-exon skipping. More importantly, because of the remarkable conservation of known splicing factors between Drosophila and humans, the information gained from these studies will significantly advance our understanding of regulated splicing in vertebrates.