INVOLVEMENT OF PROTEINS IN SPLICING GROUP I INTRONS

蛋白质参与剪接 I 组内含子

• 批准号: 2693230
• 负责人: ALAN M. LAMBOWITZ
• 金额: $ 35.9万
• 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/2693230
• 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.

拟议的研究是对参与的持续研究 脉孢菌 CYT-18 蛋白，线粒体酪氨酰-tRNA 合成酶 (mt TyrRS) 和 I 组内含子剪接中的其他蛋白质。 第一组 内含子使用RNA催化的剪接反应，但需要蛋白质 体内有效剪接，可能是为了促进正确的 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组件 脉孢菌线粒体。 在当前的资助期内，我们表明 CYT-18 蛋白（mt TyrRS）本身足以剪接基团 I 体外内含子，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 核心处于催化活性所需的构象。 我们还获得了 有证据表明 CYT-18 可以替代 RNA 结构 P5a,b,c, 四膜虫 rRNA 内含子自我剪接所必需的，我们 表明CYT-18在生理条件下可以促进反向剪接 相关条件，从而可能有助于内含子 换位。 cyt-19 成分有助于高效剪接 在体内，可能是通过帮助折叠前体 RNA，而 CYT-4 蛋白质与细胞中涉及的基因产物具有显着的相似性 循环蛋白磷酸酶具有功能并且可能具有调节作用。 研究结果 对于 CYT-18 表明 I 组内含子催化核心可能具有 类似于 tRNA 的结构特征，这可以反映 趋同进化或第一组之间的进化关系 内含子和 tRNA。 CYT-4 的发现提出了 RNA 的可能性 催化剪接反应受蛋白质磷酸化调节。 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，程度 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组内含子以及 tRNA酪氨酸。 这些研究将包括与托马斯博士的合作 Steitz（耶鲁大学）确定 CYT-18/RNA 复合物的结构。 (2) 研究蛋白质辅助的进化和后果 I组内含子的剪接。 最初的目标是阐明 CYT-18依赖基团缺乏自剪接的结构基础 I 神经孢菌线粒体中的内含子并研究 TyrRS 如何 适应拼接功能。 长期目标是 研究其他蛋白质对剪接功能的适应性， 详细分析 CYT-18 和 P5a、b、c 的功能等价性 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 在大肠杆菌中。 (3) 鉴定cyt-19成分，确定其在细胞因子中的作用 剪接，以及它是否是 CYT-4 介导的调控途径的目标 涉及蛋白质磷酸化。 该研究旨在提供 关于催化活性 RNA 相互作用的新信息 催化活性所需的蛋白质及其进化 内含子和剪接机制，这对于 真核生物。