Roles of S. pombe SR proteins in spliceosome function and assembly

描述(申请人提供)：人类大多数基因表达的一个基本步骤是去除内含子，内含子被大而动态的剪接体复合体从前-信使核糖核酸中剪接出来。已有研究表明，大多数人类遗传性疾病是由内含子的错误移除引起的。人类中的SR蛋白是重要的剪接因子，已被证明是剪接体功能和调控所必需的。更具体地说，它们已被证明在剪接体组装以及剪接反应的后期步骤中发挥重要作用。SR蛋白水平的改变已被证明会导致许多人类疾病，如癌症和艾滋病毒。考虑到9种蛋白SR家族、组织特异性表达水平和在选择性剪接中的复杂作用，在人类中区分单个SR蛋白的作用被证明是困难的。为了区分全基因组SR蛋白在遗传易控系统中的作用，我们将使用单细胞模式生物--裂殖酵母。在S.pombe中只有两个SR蛋白，其中许多内含子具有与人类相似的特征。鉴于S.pombe中许多内含子的不同内含子结构，只有几个内含子被测试依赖SR蛋白进行最佳剪接。此外，许多已被证明与人SR蛋白相互作用的剪接因子也存在于S.pombe中，尽管只观察到其中的一小部分相互作用，目前缺乏全面的分析。利用酵母菌独有的基因技术以及从人类实验中改编的生化分析，拟议的研究将确定SR蛋白如何与S.pombe(AIM1)中的剪接体相互作用，以及它们在有效去除该模式生物(AIM2)中存在的许多独特内含子的全基因组中扮演什么角色。此外，拟议的研究将通过分析剪接体在特定内含子(AIM3)上的组装来确定SR蛋白如何影响S.pombe中剪接的机制。对SR蛋白如何与S.pombe中不同的内含子一起发挥作用的基本了解将有助于了解SR蛋白的一般机制以及剪接体作为一个整体处理前mRNA的基本过程。

DESCRIPTION (provided by applicant): An essential step in the expression of the majority of genes in humans is the removal of introns, which are spliced out of pre-mRNA by the large and dynamic spliceosome complex. It has been indicated that a significant majority of hereditary disease in humans is caused by the incorrect removal of introns. SR proteins in humans are important splicing factors that have been shown to be essential for spliceosome function and regulation. More specifically, they have been shown to play essential roles in spliceosome assembly as well as later steps of the splicing reaction. Altered levels of SR proteins have been shown to result in many human diseases such as cancer and HIV. Distinguishing the roles of individual SR proteins has proven difficult in humans given the nine-protein SR family, tissue specific expression levels, and complex roles in alternative splicing. In order to distinguish the roles of SR proteins genome-wide in a genetically tractable system, the unicellular model organism Schizosaccharomyces pombe will be utilized. There are only two SR proteins in S. pombe with many introns containing similar characteristics with humans. Given the diverse intron structure of the many introns in S. pombe only a few introns have been tested for reliance on SR proteins for optimal splicing. In addition, many of the splicing factors that have been shown to interact with human SR proteins are also present in S. pombe although only a few of these interactions have been observed and a thorough analysis is currently lacking. Using genetic techniques that are uniquely available to yeast as well as biochemical assays adapted from experiments in humans, proposed studies will determine how SR proteins interact with the spliceosome in S. pombe (AIM1) and what role they play genome-wide in the efficient removal of the many unique introns that are present in this model organism (AIM2). In addition, proposed studies will determine the mechanism of how SR proteins influence splicing in S. pombe by analyzing spliceosome assembly on specific introns (AIM3). The fundamental understanding of how SR proteins function with the diverse set of introns in S. pombe will inform general mechanisms of SR proteins and the essential process of pre-mRNA processing by the spliceosome as a whole.