Mechanisms of Spliceosome Assembly and Splice Site Selection

前-信使核糖核酸剪接是真核生物基因调控的重要步骤，也是编码的中心过程 高等生物体的遗传复杂性。剪接是由一个百万吨的RNA和 被称为剪接体的蛋白质。剪接过程的关键是正确选择剪接部位 (化学位置)，以保存转录本的阅读框架和 通过选择性剪接产生合适的信使核糖核酸亚型。这个Roo应用程序的重点是使用Single 分子荧光方法研究5‘剪接位点和分支位点的识别机制 在酵母中组装剪接体时。这些机制将作为理解 人类和人类疾病中的剪接位点选择和选择性剪接。 在K99阶段开发的单分子荧光方法(参见Hoskins等，Science， 2011)允许通过遵循个体上的剪接路径来解剖复杂的反应方案 从头到尾都有前信使RNA。这些方法可直接应用于剪接位点选择的分析 在Roo阶段。5‘剪接位点最初由剪接体U1的SnRNP识别。U1 SnRNP参与许多RNA：RNA、RNA：蛋白质和蛋白质与Pre-mRNA的相互作用 所有这些合作都是为了赋予亲和力和忠诚度。利用单分子荧光，各种 这些相互作用对U1/5‘剪接位点相互作用稳定性的贡献将被量化 (具体目标1)。辅助蛋白通常有助于促进剪接体组装(例如剪接 人类中的调节蛋白)。酵母还含有促进剪接体组装的因子，而 帽结合蛋白促进减数分裂调控的前mRNAs剪接的机制将是 用单分子方法(特异靶2)进行鉴定。最后，正确选择分支机构的地点 U2SnfRNP需要通过死盒ATPase Prp5对ATP进行水解。将使用单分子方法 通过动力学校对阐明Prp5/U2/Pre-mRNA相互作用促进分支位点的保真度(特异性 目标3)。

Pre-mRNA splicing is an essential step in eukaryotic gene regulation and a central process for encoding genetic complexity in higher organisms. Splicing is carried out by a MegaDalton complex of RNA and proteins called the spliceosome. Critical to the splicing process is the correct choice of the splice sites (locations of chemistry) in the pre-mRNA in order to preserve the reading frame of the transcript and produce the proper mRNA isoform by alternative splicing. The focus of this ROO application is to use single molecule fluorescence methods to elucidate the mechanisms of 5' splice site and branchsite recognition during spliceosome assembly in yeast. These mechanisms will serve as a paradigm for understanding splice site selection and alternative splicing in humans and human disease. Single molecule fluorescence methods developed during the K99 phase (see Hoskins et al., Science, 2011) allow complex reaction schemes to be dissected by following splicing pathways on individual pre-mRNAs from start to finish. These methods can be directly applied to analysis of splice site selection during the ROO phase. The 5' splice site is initially recognized by the spliceosomal U1 snRNP. The U1 snRNP engages in a number of RNA:RNA, RNA:protein, and protein:protein interactions with the pre-mRNA that all collaborate to confer affinity and fidelity. Using single molecule fluorescence, the various contributions these interactions make to the stability of the U1/5' splice site interaction will be quantified (Specific Aim 1). Auxiliary proteins often contribute to promote spliceosome assembly (e.g. splicing regulatory proteins in humans). Yeast also contain factors that can promote spliceosome assembly, and the mechanisms by which cap binding proteins promote splicing of meiotically regulated pre-mRNAs will be elucidated with single molecule methods (Specific Aim 2). Finally, correct choice ofthe branchsite by the U2 snfRNP requires ATP hydrolysis by the DEAD-box ATPase, Prp5. Single molecufe-^methods will be used to elucidate Prp5/U2/pre-mRNA interactions that promote branchsite fidelity by kinetic proofreading (Specific Aim 3).