INVOLVEMENT OF PROTEINS IN SPLICING GROUP I INTRONS

这项拟议的研究是对 脉孢子虫Cyt-18蛋白、线粒体酪氨酸氨基-tRNA合成酶(mt TyrRS)，以及与I组内含子剪接有关的其他蛋白质。第I组 内含子使用RNA催化的剪接反应，但需要蛋白质 体内有效的剪接，可能是为了促进正确的RNA折叠。 我们确定了三个核基因，Cyt-18，Cyt-19和Cyt-4，它们编码 剪接第I组内含子所需的反式作用元件 脉孢子虫线粒体。在当前的资助期内，我们表明 细胞色素T-18蛋白，mt TyrRS，本身就足以拼接基团 在体外，I内含子识别高度保守的结构 第一类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为核心。我们还获得了 证明Cyt-18可以替代RNA结构P5a，b，c， 是四膜虫rRNA内含子自剪接所必需的，我们 证实细胞色素T-18在生理条件下可促进反向剪接 相关条件，因此有可能导致内含子 换位。Cyt-19组件有助于高效拼接 在体内，可能是通过帮助折叠前体RNA，而细胞色素T-4 蛋白质与细胞中涉及的基因产物有很大的相似性 循环蛋白磷酸酶的功能可能是调节的。调查结果 提示第一组内含子催化核心可能有 类似于tRNA中的结构特征，这可能反映 收敛进化或群I之间的进化关系 内含子和tRNA。细胞色素T-4的发现增加了RNA 催化的剪接反应受蛋白质磷酸化的调节。 具体目标是：(1)继续调查该委员会的职能 Cyt-18蛋白及其与内含子RNA和tRNatyr的相互作用 生物化学和遗传方法。我们特别感兴趣的是 细胞色素T-18如何稳定内含子核心的活性结构，程度 第一组内含子核心和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组内含子和 TRNatyr.这些研究将包括与托马斯博士的合作 Steitz(Yale)等人确定了细胞色素T-18/RNA复合体的结构。(2) 研究蛋白质辅助的进化和后果 第一组内含子的剪接。最初的目标是阐明 细胞色素T-18依赖基团缺乏自我剪接的结构基础 I内含子在脉孢菌线粒体中的表达及TyrRS如何 适用于在拼接中发挥作用。较长期的目标是 研究其他蛋白质在剪接中的功能适应性，以 详细分析细胞色素T-18与P5a、b、c的功能等价性 四膜虫内含子的结构，并建立一种 依赖细胞色素T-18的反向剪接研究内含子转座 在大肠杆菌中。(3)鉴定Cyt-19组分，确定其在 剪接，以及它是否是细胞色素T-4介导的调控途径的靶点 涉及到蛋白质的磷酸化。这项研究旨在提供 关于催化活性RNA与DNA相互作用的新信息 催化活性所需的蛋白质以及关于 内含子和剪接机制，它们在 真核生物。

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.