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

描述(由申请人提供)：拟议研究的目标是 在分裂酵母中使用遗传/基因组相结合的方法 裂殖酵母了解外显子剪接增强子的作用 以及识别它们并对它们做出反应的蛋白质。在哺乳动物身上的实验 细胞提取物导致了增强子结合的SR蛋白接触的模型 异二聚体通用剪接因子U2AF及其稳定结合 3‘端剪接位点较弱。然而，该模型与拼接的相关性 活体还没有建立起来。S.pombe是最简单的真核生物，它包含 建议的增强子复合体成分的高度保守的同源异构体。因此， 方便的遗传工具和完整的基因组序列的可用性将是 利用来扩展这些重要信号和因素的当前知识 在三个具体目标下：1)外显子剪接增强子在 促进和/或调节srp2前-mRNA的剪接，它编码 人类SRp55的裂解酵母，将使用一组 分子遗传操作之后是体内剪接的分析。b) 将寻找其他自然产生的S.pombe外显子剪接增强剂 采用基因融合策略并确定其来源基因。2)a)至 确定Srp2p是作为通用剪接因子还是作为特殊剪接因子发挥作用 在裂解酵母中，一组不同的拼接缺陷 内含子将在物理或基因耗尽蛋白质后进行分析 在活体内。B)U2AF的两个亚基在增强子依赖的vs. 不依赖于增强子的剪接将通过分析 在含有条件性突变的细胞中选择前mRNAs。3)两者都是开放式的 并将使用定向遗传策略来识别其他基因 产物与裂解酵母SR蛋白在物理或功能上相互作用 与U2AF的两个亚基结合。它们的鉴定和表征 组件不仅应该提供有关联系人的重要新信息 在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。 总而言之，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，但导致了更全球化的 它们对细胞的影响的图片。

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.