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: 6519254
• 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/6519254
• 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 粟酒裂殖酵母了解外显子剪接增强子的作用 以及识别它们并对其做出反应的蛋白质。哺乳动物实验 细胞提取物建立了一个模型，其中增强子结合的 SR 蛋白接触 异二聚体通用剪接因子 U2AF 并稳定其结合 具有弱 3' 剪接位点。然而，该模型与拼接的相关性 vivo尚未成立。粟酒裂殖酵母是最简单的真核生物，包含 所提出的增强子复合物成分的高度保守的直系同源物。因此， 简便的遗传工具和完整的基因组序列的可用性将 用于扩展这些重要信号和因素的当前知识 根据三个具体目标：1) a) 外显子剪接增强子在 促进和/或调节 srp2 前 mRNA 的剪接，其编码 人类 SRp55 的裂变酵母对应物，将使用一组 分子遗传操作，然后进行体内剪接测定。 b) 将寻找其他天然存在的粟酒裂殖酵母外显子剪接增强剂 使用基因融合策略并鉴定其源基因。 2) a) 至 确定 Srp2p 是作为通用剪接因子还是专用剪接因子 在裂变酵母中，不同组的剪接缺陷模式 内含子将在物理或遗传耗尽蛋白质后进行分析 体内。 b) U2AF 的两个亚基在增强子依赖性与非增强子依赖性中的作用 增强子独立的剪接将通过分析剪接来确定 在含有条件突变的细胞中选择前体mRNA。 3）都是开放式的 并将采用定向遗传策略来识别其他基因 产品在物理或功能上与裂殖酵母 SR 蛋白相互作用， 具有 U2AF 的两个亚基。这些的识别和表征 组件不仅应该提供有关联系人的重要新信息 已知相互作用的组件之间，但揭示了协作的新因素 与细胞内的 SR 蛋白和 U2AF 结合。 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，但导致了更加全球性的 它们对细胞影响的图片。