Function of snRNP particles in pre-mRNA splicing

描述(申请人提供)：大多数哺乳动物基因的前信使核糖核酸转录本包含多个内含子，在信使核糖核酸用于蛋白质合成之前，必须精确地去除这些内含子。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能会对健康造成毁灭性的后果，约占所有已知人类遗传疾病的15%。通过更好地了解控制内含子去除的细胞规则，剪接相关疾病的诊断和治疗将受益。前信使核糖核酸剪接酶，或剪接体，是一个由70多种蛋白质和5个小核RNA组成的大型核糖核蛋白复合体。一个被广泛引用的剪接体组装、激活和拆解(或剪接体周期)模型已经从过去15年进行的体外实验结果演变而来。剪接效率的调节和可变剪接位点的选择通常可以通过在存在或不存在特定调节蛋白(或顺式作用元件)时观察到的剪接体组装的变化来解释。最近从酵母中分离到的U2、U5、U6核糖核蛋白(RNP)复合体使人想起晚期或催化后剪接体，支持这种剪接体循环模型。相反，从酵母中鉴定出似乎完全组装但不含前mRNA5-nRNP的剪接体，提出了剪接体拆解是否真的在体内发生的问题。我们将通过测试两个假设来解决这一明显的差异。首先，剪接体循环发生在细胞内，并通过一系列依赖剪接因子的组装和拆卸步骤进行。其次，Clf1p组装因子通过包括RNA输出因子招募在内的分支点重组，促进了前复制小体与U4/U6.U5三亚单位RNP颗粒的整合。这项工作将为分析RNA的加工过程提供新的实验工具，并建立概念框架，从这些框架中观察剪接体的组装以及活细胞中前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.