RNA SPLICING IN MITOCHONDRIA

这项拟议的研究是对蛋白质的生化遗传学研究。 在第一组内含子剪接中起作用的因子，使用 以脉孢子虫和酵母线粒体为实验系统。 I组线粒体(Mt)内含子属于同一结构 类作为四膜虫核rRNA内含子，并使用相同的 本质上是RNA催化的剪接机制。我们有 确定了在拼接基团方面有缺陷的核突变体 脉孢霉Mt DNA基因编码大分子Mt的I内含子 RRNA。这些突变体映射到四个不同的核基因。这个 Cyt-18和Cyt-19突变体在拼接多个 不同的I族内含子，并且可能是有缺陷的成分 通常在拼接这些内含子或某个亚类时起作用 其中之一。本资助期内的研究显示， Cyt-18突变体严重缺乏一种可溶性活性， 在剪接mt大rRNA内含子和可能的其他 第一组内含子，而Cyt-19-1突变体可能存在缺陷 这会损害这一活动与RNP的结合。克隆和 对Cyt-18基因的测序表明，它含有一个开放的 与细菌酪氨酸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酪氨酰-tRNA合成酶，即 Cyt-18基因的突变直接影响剪接，而mt. 酪氨酰-tRNA合成酶或该蛋白质的某些衍生物 与可溶性剪接活性有关。我们建议继续 学习。具体目标是：(1)确定精准 Mt酪氨酰-tRNA合成酶与剪接的关系 活动。(2)研究Cyt-18基因突变如何 影响剪接和合成酶活性。(3)识别其他 作为其组成部分的多肽或具有联合作用的多肽 具有剪接活性。(4)测定mt tyrosyl-tRNA 合成酶和/或剪接活性在I组剪接中的作用 内含子。(5)鉴定额外的核基因和基因 脉孢子菌I组内含子剪接功能的产物 线粒体。(6)在合作研究中，使用生化 类似于为脉孢菌属开发的方法 研究酵母线粒体I组内含子的剪接 初步强调mt tyrosyl-tRNA的可能参与 合成酶和其他mt氨酰-tRNA合成酶。这些 研究的目的是提供关于 催化活性RNA与所需蛋白质的相互作用 对于催化活性，并可能提供对进化的洞察 真核生物的内含子和剪接机制。

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.