SNRNP-SUBSTRATE INTERACTIONS IN S POMBE SPLICING

SNRNP-底物相互作用在 S Pombe 剪接中的作用

• 批准号: 3294082
• 负责人: JO ANN WISE
• 金额: $ 18.03万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3294082
• 关键词: RNA splicing; Schizosaccharomyces pombe; fungal genetics; genetic manipulation; messenger RNA; molecular cloning; nucleic acid sequence; small nuclear RNA; tissue /cell culture

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.

描述：（根据申请人的摘要改编）：核电站所在地 前信使 RNA 剪接是一种核糖体大小的复杂结构 称为剪接体的亚基。 它的许多组成部分仍有待 已识别，并且仅一小部分已被表征 分子水平。 虽然剪接的一般机制是保守的 通过进化，更详细的研究揭示了两者的差异 snRNA 的结构是机器的核心， 它们在基质中识别的信号等。 申请人 已开始对裂殖酵母中的前 mRNA 剪接进行分析 粟酒裂殖酵母 (Schizosaccharomyces pombe) 因其与 剪接已得到更广泛研究的生物体使得 非常适合区分所有真核生物所共有的方面 祖先机制的其他特定群体的专业化。 一个 该生物体的另一个优点是它易于遗传 通过古典和现代方法进行操纵。 申请人将 最集中地探索 U1 snRNP 功能的各个方面。 一、U1 snRNA基因的体外诱变及模型剪接 将使用底物，然后进行转化和表型分析 描述他们的互动；除了提供系统发育 对先前观察到的 5' 连接识别差异的看法 研究人员，这些研究将测试申请人拥有的模型 为 U1 与 3' 连接的氢键结合而开发。 一个开放式的 诱变策略将使研究人员能够识别 U1 snRNA 蛋白对于剪接体组装以及其他可能很重要， 尚未被怀疑的功能。 抑制无法拼接的缺陷 被 snRNA 中的补偿性突变完全拯救将被用于 识别与剪接信号相互作用的非 snRNP 因子。 中学 该计划的目标将是检查进化起源和 U6 snRNA基因内含子的功能意义，以及 U5 snRNA 与 5' 剪接潜在相互作用的意义 地点。 根据体内实验确定的相互作用将 使用同源体外剪接提取物进行详细检查，这将 让我们能够观察剪接体组装的中间步骤，以便 确定突变表型的生化基础。