In Vivo Analysis of Spliceosomal Protein Function

剪接体蛋白功能的体内分析

• 批准号: 6889882
• 负责人: HELEN Karen SALZ
• 金额: $ 28.92万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6889882
• 关键词: Drosophilidae; RNA splicing; gene interaction; male; male reproductive system; northern blottings; nucleic acid sequence; polymerase chain reaction; protein protein interaction; protein structure function; small nuclear ribonucleoproteins; spliceosomes; yeast two hybrid system

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.

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的体内功能 规定。以性别特异性剪接调控为例 果蝇二元开关基因Sex-lethal (Sxl)，研究表明 SANS-FILLE (SNF), 人类U1 snRNP-U1A和U2 snRNP-U2B的对应物" 蛋白质，在 Sxl 剪接自动调节中起着关键作用。虽然很清楚 SNF 和女性特有的 SXL 蛋白发生物理相互作用并被发现 在同一个多组分复合体中，该复合体中其他成员的身份 复杂以及它们如何组装以抑制雄性特异性的剪接 外显子仍有待确定。 为了解决这些问题，提出了三个具体目标。在目标 1 中， 假设，基于遗传和蛋白质-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 通过包括蛋白质定位研究的组合方法进行测试， 蛋白质-蛋白质相互作用研究以及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 将通过利用研究中的信息来识别自动调节 酵母和哺乳动物细胞在直系同源剪接因子中产生突变 并测试它们的遗传相互作用和未能持续跳过 Sxl 男性外显子。尚未连接剪接的候选位点 将通过全基因组遗传筛查以及生化检测来鉴定 确认。在目标 3 中，SNF 及其伙伴的生化特性 对女性特异性 Sxl 剪接重要的蛋白质将使用以下方法进行鉴定 蛋白质-蛋白质和蛋白质-RNA 相互作用测定。此外，分子 控制男性外显子利用的途径将通过确定来阐明 哪些相互作用取决于 SXL 和/或关联的存在 使用突变动物的提取物在 SXL 和 SNF 之间进行比较。 这三个目标的综合数据将为 SXL 如何、 SNF 及其蛋白质伙伴被组装成阻断复合物并 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着保守性 人类，从这些研究中获得的信息将显着推进 我们对脊椎动物调控剪接的理解。