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: 2179118
• 负责人: JO ANN WISE
• 金额: $ 18.11万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 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 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179118
• 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：(适用于申请者摘要)：核设施所在地 前信使RNA剪接是一个核糖体大小的复杂结构 被称为剪接体的亚基。它的许多组件仍有待于 已确定，并且只有一小部分在 分子水平。而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之间的差异 位于机器核心的小分子RNA的结构 他们在底物中识别的信号，以及其他。申请人 已经开始分析裂解酵母中的前-mRNA剪接 庞氏裂殖酵母是因为它与裂殖酵母广泛的系统发育 对剪接进行了更广泛研究的生物体使其 非常适合区分所有真核生物共有的方面 其他特定于群体的祖先机制的专门化。一个 这种生物的另一个优势是它能适应遗传 通过古典和现代两种方法进行操纵。申请者将会 最专注于探索U1 snRNP功能的各个方面。 首先，U1单链RNA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 来描述它们之间的相互作用；除了提供系统发育 前人观察到的5‘连接识别差异透视 调查人员，这些研究将测试申请者的一个模型 为U1与3‘结的氢键而开发。不限成员名额的 诱变策略将允许调查者识别残留物 U1 SnRNA蛋白对剪接体组装和其他可能是重要的， 到目前为止，还没有人怀疑它起作用了。抑制不能实现的拼接缺陷 通过SNRNA的补偿性突变完全挽救将被用来 识别与剪接信号相互作用的非SNRNP因子。次要的 该计划的目标将是研究进化的起源和 U6单链RNA基因内含子的功能意义 U5单链RNA与5‘端剪接体潜在相互作用的意义 地点。根据活体实验确定的相互作用将是 使用同源的体外剪接提取物进行了详细的检查，这将 允许我们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。