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 Neurospora Cyt-18蛋白，线粒体酪酶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 剪接组I内含子所需的跨作用组件 神经孢子线粒体。 在当前赠款期间，我们表明 Cyt-18蛋白MT Tyrr本身足以拼接组 我的体外内含子，它识别高度保守的结构 组I内含子催化核心的功能，并稳定 催化活性所需的构象中的核心。 我们也获得了 Cyt-18可以代替RNA结构P5A，B，C， 四心肌rRNA内含子的自剪接所必需的，我们 鞋子表明Cyt-18可以在生理上促进反向剪接 相关条件，因此有可能有助于内含子 换位。 CYT-19组件有助于有效的剪接 在体内，可能是通过帮助折叠前体RNA，而Cyt-4 蛋白质与涉及细胞的基因产物具有显着相似性 循环蛋白磷酸酶的功能，可能是调节性的。 发现 对于Cyt-18，表明I组I内含子催化核可能具有 类似于trnas的结构特征，可以反映 收敛进化或I组之间的​​进化关系 内含子和trnas。 CYT-4的发现提高了RNA的可能性 催化的剪接反应受蛋白质磷酸化调节。 具体目的是：（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 生化和遗传方法。 我们对 Cyt-18如何稳定内含子芯的活动结构 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绑定I组内含子和 trnatyr。 这些研究将包括与托马斯博士的合作 Steitz（YALE）确定Cyt-18/RNA复合物的结构。 （2） 研究蛋白质辅助的演变和后果 I组内含子的剪接。 最初的目标是阐明 缺乏CYT-18依赖性组自剪接的结构基础 I Neurospora线粒体中的内含子，并研究Tyrrs如何 适应剪接功能。 长期目标是 研究其他蛋白质在剪接中起作用的适应 详细分析CYT-18和P5A，B，C的功能当量 四系内含子的结构，并建立一个系统 通过CYT-18依赖性反拼接研究内含子换位 在大肠杆菌中。 （3）确定CYT-19组件，确定其在 剪接，如果是Cyt-4介导的调节途径的目标 涉及蛋白质磷酸化。 该研究旨在提供 有关催化活性RNA与 催化活性所需的蛋白质以及关于的演变 内含子和剪接机制，在根本上很重要 真核生物。