Pre-mRNA Splicing in S. pombe: Genetics and Genomics

粟酒裂殖酵母中的前 mRNA 剪接：遗传学和基因组学

• 批准号: 6398851
• 负责人: JO ANN WISE
• 金额: $ 30.98万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6398851
• 关键词: RNA splicing; Schizosaccharomyces pombe; complementary DNA; functional /structural genomics; fungal genetics; fungal proteins; gene induction /repression; genetic library; intermolecular interaction; introns; microarray technology; molecular site; nucleic acid sequence; polymerase chain reaction; precursor mRNA; protein structure function; reporter genes; ribonucleoproteins; site directed mutagenesis; small nuclear RNA; spliceosomes

DESCRIPTION (provided by applicant): The goal of the proposed research is to use a combined genetic/genomic approach in the fission yeast Schizosaccharomyces pombe to understand the roles of exonic splicing enhancers and the proteins that recognize and respond to them. Experiments in mammalian cell extracts have led to a model in which enhancer-bound SR proteins contact the heterodimeric general splicing factor U2AF and stabilize its association with weak 3' splice sites. However, the relevance of this model to splicing in vivo has not been established. S. pombe is the simplest eukaryote that contains highly conserved orthologues of proposed enhancer complex constituents. Thus, the availability of facile genetic tools and a complete genome sequence will be exploited to extend current knowledge of these important signals and factors under three specific aims: 1) a) The role of an exonic splicing enhancer in promoting and/or regulating splicing of srp2 pre-mRNA, which encodes the fission yeast counterpart of human SRp55, will be delineated using a battery of molecular genetic manipulations followed by assays of splicing in vivo. b) Other naturally occurring S. pombe exonic splicing enhancers will be sought using a gene fusion strategy and their source genes identified. 2) a) To determine whether Srp2p functions as a general or specialized splicing factor in fission yeast, the pattern of splicing defects for a diverse panel of introns will be analyzed after physically or genetically depleting the protein in vivo. b) The roles of both subunits of U2AF in enhancer-dependent vs. enhancer-independent splicing will be determined by assaying splicing of selected pre-mRNAs in cells harboring conditional mutations. 3) Both open-ended and directed genetic strategies will be employed to identify other genes whose products physically or functionally interact with fission yeast SR proteins and with the two subunits of U2AF. Identification and characterization of these components should not only provide important new information about contacts between components known to interact, but reveal novel factors that collaborate with SR proteins and U2AF within the cell. Together, these studies should not only shed new light on specific questions about the early events of pre-messenger RNA splicing, but lead to a more global picture of their impact on the cell.

描述（由申请人提供）：拟议研究的目标是 在裂殖酵母中使用组合遗传/基因组方法 粟酒裂殖酵母，以了解外显子剪接增强子的作用 以及识别和应答它们的蛋白质。哺乳动物实验 细胞提取物导致了一种模型，其中增强子结合的SR蛋白接触 异二聚体通用剪接因子U2AF及其稳定缔合 具有弱的3 '剪接位点。然而，该模型与剪接的相关性 vivo尚未建立。S.粟酒属是最简单的真核生物， 提出的增强子复合物组分的高度保守的直向同源物。因此，在本发明中， 容易获得遗传工具和完整的基因组序列将 用于扩展这些重要信号和因素的现有知识 在三个具体目标下：1）a）外显子剪接增强子在 促进和/或调节srp 2前mRNA的剪接，该前mRNA编码 人SRp55的分裂酵母对应物，将使用一组 分子遗传操作，随后进行体内剪接测定。B） 自然发生的S。将寻找粟酒属外显子剪接增强子 使用基因融合策略并鉴定它们的源基因。2)（a） 确定p2p是作为一般的还是专门的剪接因子起作用 在裂殖酵母中，不同的基因组的剪接缺陷模式 内含子将在物理上或遗传上耗尽蛋白质后进行分析 in vivo. B）U2 AF的两个亚基在增强子依赖性vs. 增强子非依赖性剪接将通过测定 在携带条件突变的细胞中选择前mRNA。3)都是开放式的 和定向遗传策略将被用来识别其他基因， 产物在物理上或功能上与裂殖酵母SR蛋白相互作用， 与U2AF的两个亚基结合。鉴定和表征这些 组件不仅应提供有关联系人的重要新信息， 在已知的相互作用的组件之间，但揭示了新的因素， 与SR蛋白和U2AF在细胞内。 总之，这些研究不仅应该为具体问题提供新的线索， 关于前信使RNA剪接的早期事件，但导致了一个更全球性的 它们对细胞的影响。