INVOLVEMENT OF PROTEINS IN SPLICING GROUP I INTRONS

蛋白质参与剪接 I 组内含子

• 批准号: 2900641
• 负责人: ALAN M. LAMBOWITZ
• 金额: $ 36.78万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900641
• 关键词: Neurospora; RNA splicing; aminoacid tRNA ligase; binding proteins; conformation; enzyme activity; enzyme mechanism; eukaryote; fungal genetics; gel electrophoresis; gene expression; genetic mapping; introns; mitochondria; molecular cloning; nucleic acid hybridization; nucleic acid sequence; open reading frames; phosphorylation; protein folding; radiotracer; regulatory gene; ribosomal RNA

The proposed research is a continued study of the involvement of the Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase (mt TyrRS), and other proteins in the splicing of group I introns. Group I introns use RNA catalyzed splicing reactions, but require proteins for efficient splicing in vivo, presumably to facilitate correct RNA folding. We identified three nuclear genes, cyt-18, cyt-19, and cyt-4, that encode trans-acting components required for splicing group I introns in Neurospora mitochondria. During the current grant period, we showed that the CYT-18 protein, the mt TyrRS, is itself sufficient to splice group I introns in vitro, that it recognizes highly conserved structural features of the group I intron catalytic core, and that it stabilizes the core in a conformation required for catalytic activity. We also obtained evidence that CYT-18 can substitute for an RNA structure, P5a,b,c, required for the self-splicing of the Tetrahymena rRNA intron, and we shoed that CYT-18 could promote reverse splicing under physiologically relevant conditions and thus potentially contribute to intron transposition. the cyt-19 component contributes to efficient splicing in vivo, probably by helping fold the precursor RNA, whereas the CYT-4 protein has significant similarity to gene products involved in cell cycle protein phosphatase functions and may be regulatory. The findings for CYT-18 suggest that the group I intron catalytic core may have structural features that resemble those in tRNAs, which could reflect convergent evolution or an evolutionary relationship between group I introns and tRNAs. The findings for CYT-4 raise the possibility that RNA catalyzed splicing reactions are regulated by protein phosphorylation. Specific aims are: (1) To continue to investigate the function of the CYT-18 protein and its interaction with the intron RNA and tRNATyr, using biochemical and genetic approaches. We are particularly interested in how CYT-18 stabilizes the active structure of the intron core, the extent of structural similarity between the group I intron core and tRNAs, and whether CYT-18 uses similar interactions to bind group I introns and tRNATyr. These studies would include a collaboration with Dr. Thomas Steitz (Yale) to determine the structures of CYT-18/RNA complexes. (2) To investigate the evolution and consequences of protein-assisted splicing of group I introns. Initial objectives are to elucidate the structural basis for the lack of self-splicing of CYT-18-dependent group I introns in Neurospora mitochondria and to investigate how the TyrRS adapts to function in splicing. Longer term objectives are to investigate the adaptation of other proteins to function in splicing, to analyze in detail the functional equivalence of CYT-18 and the P5a,b,c structure of the Tetrahymena intron, and to establish a system for investigating intron transposition via CYT-18-dependent reverse splicing in E. coli. (3) To identify the cyt-19 component, determine its role in splicing, and if it is the target of a CYT-4-mediated regulatory pathway involving protein phosphorylation. The research is intended to provide novel information about the interaction of catalytically active RNAs with proteins required for catalytic activity and about the evolution of introns and splicing mechanisms, which are fundamentally important in eukaryotes.

拟议的研究是一项持续的研究， 链孢菌CYT-18蛋白，线粒体酪氨酰-tRNA合成酶（mt TyrRS）和其它蛋白质在I组内含子剪接中的作用。 组I 内含子使用RNA催化的剪接反应，但需要蛋白质 在体内有效剪接，大概是为了促进正确的RNA折叠。 我们鉴定了三个核基因，cyt-18，cyt-19和cyt-4，它们编码 剪接I组内含子所需的反式作用元件 线粒体脉孢菌。 在目前的赠款期间，我们表明， CYT-18蛋白，mt TyrRS，本身足以剪接基团， I内含子，它识别高度保守的结构 I组内含子催化核心的特征，并且它稳定了 以催化活性所需的构象形成核心。 我们也得到 CYT-18可以取代RNA结构P5 a，B，c， 四膜虫rRNA内含子的自我剪接所需的，我们 提示CYT-18在生理条件下可促进反向剪接 相关的条件，从而可能有助于内含子 换位 CYT-19组分有助于有效剪接 在体内，可能是通过帮助折叠前体RNA，而CYT-4 蛋白质与细胞中的基因产物具有显著的相似性， 循环蛋白磷酸酶的功能和可能是调节。 这些发现 对于CYT-18，表明I组内含子催化核心可能具有 结构特征与tRNA相似，这可能反映了 趋同进化或第一类之间的进化关系 内含子和tRNA。 CYT-4的发现提高了RNA 催化的剪接反应由蛋白质磷酸化调节。 具体目标是：（1）继续研究 CYT-18蛋白及其与内含子RNA和tRNATyr的相互作用，使用 生物化学和遗传学方法。 我们特别感兴趣的是 CYT-18如何稳定内含子核心的活性结构， I组内含子核心和tRNA之间的结构相似性，以及 CYT-18是否使用类似的相互作用来结合I组内含子， tRNATyr. 这些研究将包括与托马斯博士的合作 Steitz（Yale）来确定CYT-18/RNA复合物的结构。 （二） 为了研究蛋白质辅助的进化和后果， I组内含子的剪接。 初步目标是阐明 CYT-18依赖性基团缺乏自我剪接的结构基础 脉孢菌线粒体中的I内含子并研究TyrRS如何 适应于拼接功能。 长期目标是 研究其他蛋白质在剪接中的适应功能， 详细分析CYT-18与P5 a、B、c的功能等效性 四膜虫内含子的结构，并建立一个系统， 通过CYT-18依赖性反向剪接研究内含子转座 在大肠杆菌 (3)为了鉴定cyt-19组分，确定其在 剪接，如果它是CYT-4介导的调控途径的靶点， 涉及蛋白质磷酸化。 该研究旨在提供 关于催化活性RNA与 蛋白质所需的催化活性和有关的演变 内含子和剪接机制，这是至关重要的， 真核生物