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）：拟议研究的目的是 描述参与剪接的蛋白质体内功能 规定。以性别特定的剪接调节为例 果蝇二进制开关基因性致命（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剪接自动调节中起关键作用。虽然很清楚 SNF和女性特异性SXL蛋白在物理上相互作用，并被发现 在同一多组分复合物中，其他成员的身份 复合物以及如何组装以抑制男性特异性的剪接 外显子仍然有待确定。 为了解决这些问题，提出了三个具体目标。在AIM 1中 基于遗传和蛋白质蛋白质相互作用研究的数据，假设， SPP-87B和SIN在SXL剪接自动调节中扮演不可或缺的角色将是 通过一种将包括蛋白质定位研究的组合方法测试， 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AIM 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从研究中的研究来确定 酵母和哺乳动物细胞在直系同源剪接因子中产生突变 并测试它们是否既有遗传相互作用又无法持续跳过 SXL雄性外年。尚未连接到剪接的候选基因座 将在全基因组遗传筛选中鉴定出与生化 确认。在AIM 3中，SNF及其合作伙伴的生化特性 使用对女性特异性SXL剪接重要的蛋白质将使用 蛋白质 - 蛋白质和蛋白-RNA相互作用测定。另外，分子 通过识别，控制男性外观利用的途径将阐明 哪些相互作用取决于SXL和/或关联的存在 在SXL和SNF之间使用突变动物的提取物。 这三个目标的合并数据将为SXL如何提供新的见解。 SNF及其蛋白质伴侣被组装成一个阻滞复合物， 共同发挥作用，以促进男性跳过。更重要的是，因为 果蝇和 人类，从这些研究中获得的信息将大大推进 我们对脊椎动物中受调节的剪接的理解。