RNA SPLICING IN MITOCHONDRIA

拟议的研究是蛋白质的生化遗传学研究 在I组内含子的剪接中起作用的因子，使用 以脉孢菌和酵母线粒体为实验体系。 组I线粒体（mt）内含子属于相同的结构 类为四膜虫核rRNA内含子，并使用相同的 RNA催化的剪接机制。 我们有 确定了在剪接组中有缺陷的核突变体， 链孢菌线粒体DNA基因中的I内含子编码线粒体大 rRNA。 这些突变体映射到四个不同的核基因。 的 cyt-18和cyt-19突变体在剪接许多 不同的I组内含子，并且可能在 通常在剪接这些内含子或某些亚类中起作用 它们的 本赠款期内的研究表明， Cyt-18突变体严重缺乏可溶性活性， 在剪接mt大rRNA内含子和可能的其他功能中起作用 I组内含子，而cyt-19-1突变体可能有缺陷， 削弱这种活性与RNP的结合。 克隆及 对cyt-18基因的测序表明，它含有一个开放的 与细菌酪氨酰具有显著同源性的阅读框 tRNA合成酶。 生化和遗传实验表明 cyt-18基因编码mt酪氨酸-tRNA合成酶， cyt-18基因的突变直接影响剪接， 酪氨酰-tRNA合成酶或该蛋白的某些衍生物， 与可溶性剪接活性有关。 我们建议继续 问题研究 具体目标是：（1）确定精确的 线粒体酪氨酰-tRNA合成酶与剪接的关系 活动 (2)为了研究cyt-18基因的突变 影响剪接和合成酶活性。 (3)物色其他 作为结合蛋白的成分或功能的多肽 具有剪接活性。 (4)为了确定mt酪氨酰-tRNA 合成酶和/或剪接活性在组I剪接中起作用 内含子 (5)为了表征额外的核基因和基因 在链孢菌中剪接I组内含子的产物 线粒体 (6)在合作研究中，使用生物化学 类似于为脉孢菌开发的方法， 研究酵母线粒体中I组内含子剪接， 初步强调可能涉及mt酪氨酰-tRNA 合成酶和其它mt氨酰-tRNA合成酶。 这些 研究的目的是提供有关 催化活性RNA与所需蛋白质的相互作用 催化活性，并可能提供深入了解的演变 内含子和剪接机制。

The proposed research is a biochemical-genetic study of protein factors that function in splicing of group I introns, using Neurospora and yeast mitochondria as the experimental systems. Group I mitochondrial (mt) introns belong to the same structural class as the Tetrahymena nuclear rRNA intron and use the same essentially RNA catalyzed splicing mechanism. We have identified nuclear mutants that are defective in splicing the group I intron in the Neurospora mt DNA gene encoding the mt large rRNA. These mutants map to four different nuclear genes. The cyt-18 and cyt-19 mutants are defective in splicing a number of different group I introns and may be defective in components that function generally in splicing these introns or some subclass thereof. Studies during the current grant period showed that the cyt-18 mutants are grossly deficient in a soluble activity that functions in splicing the mt large rRNA intron and possibly other group I introns, whereas the cyt-19-1 mutant may have a defect that impairs binding of this activity to RNPs. Cloning and sequencing of the cyt-18 gene showed that it contains an open reading frame having significant homology to bacterial tyrosyl- tRNA synthetases. Biochemical and genetic experiments indicate that the cyt-18 gene encodes mt tyrosyl-tRNA synthetase, that mutations in the cyt-18 gene affect splicing directly, and that mt tyrosyl-tRNA synthetase or some derivative of this protein is related to the soluble splicing activity. We propose to continue studies. Specific aims are: (1) To determine the precise relationship between mt tyrosyl-tRNA synthetase and splicing activity. (2) To investigate how mutations in the cyt-18 gene affect splicing and synthetase activity. (3) To identify other polypeptides that are constituents of or function in conjunction with splicing activity. (4) To determine how mt tyrosyl-tRNA synthetase and/or splicing activity function in splicing of group I introns. (5) To characterize additional nuclear genes and gene products that function in splicing group I introns in Neurospora mitochondria. (6) In collaborative studies, to use biochemical approaches similar to those developed for Neurospora to investigate splicing of group I introns in yeast mitochondria, with initial emphasis on the possible involvement of mt tyrosyl-tRNA synthetase and other mt aminoacyl-tRNA synthetases. These studies are intended to provide basic information about the interaction of catalytically active RNAs with proteins required for catalytic activity and may provide insight into the evolution of introns and splicing mechanisms in eukaryotes.