SYSTEMATIC TWO-HYBRID AND COMPARATIVE PROTEOMIC ANALYSES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 Prp 19是NineTeen复合物或NTC的创始成员，NTC是剪接体激活所必需的剪接体亚复合物。为了定义Prp 19连接和动态蛋白质相互作用的剪接体内，我们系统地查询酿酒酵母蛋白质组的Prp 19 WD 40域相互作用的合作伙伴，通过双杂交分析。我们报告说，除了S。在酿酒酵母Cwc 2中，剪接因子Prp 17以1：1的比例直接结合Prp 19 WD 40结构域。Prp 17与Cwc 2同时结合，表明它是核心NTC复合物的一部分。我们还发现，以前未表征的蛋白Urn 1（Dre 4在粟酒裂殖酵母）直接与Prp 19相互作用，Dre 4是有条件地需要在S前mRNA剪接。粟酒S. pombe Dre 4和S.酿酒酵母Urn 1共纯化U2、U 5和U6 snRNA和多种剪接因子，dre 4和urn 1菌株显示出与已知剪接突变体的许多负遗传相互作用。色葡萄含有Pombe Prp 19的Dre 4复合物共纯化了三种以前未表征的参与前mRNA剪接的蛋白质，可能在剪接体激活之前。我们的多方面的方法揭示了新的低丰度剪接因子连接到NTC功能，提供了证据，不同的Prp 19含有复合物，并强调了Prp 19 WD 40域作为剪接支架的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1:1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe) directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold.