SNRNP-SUBSTRATE INTERACTIONS IN S POMBE SPLICING

描述：（适用于申请人摘要）：核试验地点 前信使RNA剪接是一种复杂的结构， 称为剪接体的亚基。 它的许多组成部分仍然是 只有一个小的子集已经确定， 分子水平。 虽然剪接的一般机制是保守的， 通过进化，更详细的研究表明， snRNA的结构位于机器的核心， 它们在基质中识别的信号等等。 申请人 已经开始分析分裂酵母中的前mRNA剪接 粟酒裂殖酵母，因为它的巨大的系统发育分离， 剪接已被广泛研究的生物体， 理想的区分这些方面普遍适用于所有真核生物， 其他的，群体特异性的，祖先机制的专门化。 一个 这种生物的另一个优点是它对遗传的适应性。 通过古典和现代的方法进行操作。 申请人将 重点集中在探索U1 snRNP功能的各个方面。 首先，U1 snRNA基因的体外诱变和模型剪接 将使用底物，然后进行转化和表型分析 描述它们的相互作用;除了提供系统发育 以前观察到的5'连接识别差异的观点 调查人员，这些研究将测试申请人拥有的模型， 开发用于U1与3'结的氢键合。 不限成员名额 诱变策略将允许研究者鉴定 U1 snRNA蛋白质对剪接体组装很重要， 尚未被怀疑的功能。 抑制剪接缺陷， 通过snRNA中的补偿突变完全获救将用于 鉴定与剪接信号相互作用的非snRNP因子。 二次 该计划的目标将是研究进化的起源， U6 snRNA基因中内含子的功能意义以及 U 5 snRNA与5'剪接的潜在相互作用的意义 绝佳的价钱 根据体内实验确定的相互作用将 使用同源的体外剪接提取物详细检查， 允许我们观察剪接体组装中的中间步骤， 确定突变表型的生化基础。

DESCRIPTION: (Adapted for applicant's abstract): The site of nuclear pre-messenger RNA splicing is a complex structure the size of a ribosomal subunit known as the spliceosome. Many of its components remain to be identified, and only a small subset have been characterized at the molecular level. While the general mechanism of splicing is conserved through evolution, more detailed studies have revealed differences both in the structures of the snRNAs which lie at the heart of the machinery and in the signals they recognize in the substrate, among others. The applicant has embarked on an analysis of pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe because its vast phylogenetic separation from organisms in which splicing has been more extensively studied makes it ideal for distinguishing those facets universal to all eukaryotes from other, group-specific, specializations of the ancestral mechanism. An additional advantage of this organism is its amenability to genetic manipulation both by classical and modern methods. The applicant will focus most intensively on exploring all aspects of U1 snRNP function. First, in vitro mutagenesis of the U1 snRNA gene and model splicing substrates followed by transformation and phenotypic analysis will be used to characterize their interactions; in addition to providing phylogenetic perspective on differences in 5' junction recognition observed by previous investigators, these studies will test a model the applicants have developed for hydrogen bonding of U1 to the 3' junction. An open-ended mutagenesis strategy will allow the investigator to identify residues of the U1 snRNA proteins important for spliceosome assembly and perhaps other, as yet unsuspected, functions. Suppression of splicing defects that cannot be fully rescued by compensatory mutations in an snRNA will be used to identify non-snRNP factors that interact with splicing signals. Secondary goals of the program will be to examine the evolutionary origin and functional significance of the intron in the U6 snRNA gene, and the significance of a potential interaction of U5 snRNA with the 5' splice site. Interactions identified on the basis of in vivo experiments will be examined in detail using a homologous in vitro splicing extract, which will allow us to observe intermediate steps in spliceosome assembly in order to determine the biochemical basis of mutant phenotypes.