Function of snRNP particles in pre-mRNA splicing

描述（由申请人提供）：大多数哺乳动物基因的pre-mRNA转录本含有多个内含子，必须在mRNA用于蛋白质合成之前精确去除内含子。对于许多基因来说，这种前mrna剪接反应是由发育或环境信号调节的。剪接途径中的错误可造成毁灭性的健康后果，约占所有已知人类遗传疾病的15%。剪接相关疾病的诊断和治疗将受益于更好地理解控制内含子移除的细胞规则。前mrna剪接酶或剪接体是由70多种蛋白质和5个小核rna组成的大型核糖核蛋白复合物。一个被广泛引用的剪接体组装、激活和拆卸（或剪接体周期）模型是从过去15年进行的体外实验结果演变而来的。剪接效率的调节和选择性剪接位点的选择通常可以通过剪接体在存在或不存在特定调节蛋白（或顺式作用元件）时观察到的组装变化来解释。最近从酵母中分离出的U2，U5，U6核糖核蛋白（RNP）复合体使人联想到后期或催化后剪接体，支持这种剪接体循环模型。相比之下，从酵母中鉴定出的似乎是完全组装但不含mrna的“penta-snRNP”剪接体提出了剪接体是否在体内实际发生拆卸的问题。我们将通过对两个假设的检验来解决这个明显的差异。首先，剪接体周期发生在细胞内，并通过一系列剪接因子依赖的组装和拆卸步骤进行。其次，Clf1p组装因子促进了前质体与U4/U6的集成。U5 tri-snRNP粒子通过分支点重组包括RNA输出因子的募集。这项工作将为分析RNA加工提供新的实验工具，并产生概念框架，从中观察剪接体组装以及活细胞中pre-mRNA剪接和RNA输出途径的整合。

DESCRIPTION (provided by applicant): The pre-mRNA transcripts of most mammalian genes contain multiple introns that must be precisely removed before the mRNA can be used in protein synthesis. For many genes, this pre-mRNA splicing reaction is regulated by developmental or environmental signals. Errors in the splicing pathway can have devastating health consequences and account for approximately 15% of all known human genetic disorders. The diagnosis and treatment of splicing-related disorders will profit by a better understanding of the cellular rules governing intron removal. The pre-mRNA splicing enzyme, or spliceosome, is a large ribonucleoprotein complex composed of over 70 proteins and 5 small nuclear RNAs. A widely cited model of spliceosome assembly, activation, and disassembly (or the spliceosome cycle) has evolved from the results of in vitro experimentation conducted over the last 15 years. The modulation of splicing efficiency and alterative splice site selection often can be explained by changes in spliceosome assembly observed in the presence or absence of specific regulator proteins (or cis-acting elements). The recent isolation from yeast of a U2,U5,U6 ribonucleoprotein (RNP) complex reminiscent of a late stage or post-catalytic spliceosome supports this spliceosome cycle model. In contrast, the identification of what appears to be a fully assembled but pre-mRNA-free "penta-snRNP" spliceosome from yeast raises the question of whether spliceosome disassembly actually occurs in vivo. We will address this apparent discrepancy by tests of two hypotheses. First, that a spliceosome cycle occurs within cells and progresses through a series of splicing factor-dependent assembly and disassembly steps. Second, that the Clf1p assembly factor facilitates integration of the prespliceosome with the U4/U6.U5 tri-snRNP particle through branchpoint reorganization that includes the recruitment of RNA export factors. This work will provide new experimental tools for the analysis of RNA processing and generate conceptual frameworks from which to view spliceosome assembly and the integration of the pre-mRNA splicing and RNA export pathways in living cells.