RNA SPLICING IN MITOCHONDRIA

线粒体中的 RNA 剪接

基本信息

批准号：
3293854
负责人：
ALAN M. LAMBOWITZ
金额：
$ 19.97万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-09-01 至 1993-03-31
项目状态：
已结题

项目摘要

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.

拟议的研究是蛋白质的生化遗传学研究在 I 组内含子剪接中起作用的因素，使用脉孢菌和酵母线粒体作为实验系统。 I 组线粒体 (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本质上是RNA催化的剪接机制。我们有鉴定出在剪接组中存在缺陷的核突变体脉孢菌 mt DNA 基因中的 I 内含子编码 mt 大核糖核酸。这些突变体映射到四种不同的核基因。这 cyt-18 和 cyt-19 突变体在剪接多个不同的 I 组内含子，并且可能在组件中有缺陷通常在剪接这些内含子或某些子类中发挥作用其中。当前资助期内的研究表明 cyt-18 突变体严重缺乏可溶性活性，剪接 mt 大 rRNA 内含子和其他可能的功能 I 组内含子，而 cyt-19-1 突变体可能有缺陷这会损害该活性与 RNP 的结合。克隆和 cyt-18基因的测序表明它含有一个开放的阅读框与细菌酪氨酰基具有显着同源性 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， cyt-18基因的突变直接影响剪接，并且mt 酪氨酰-tRNA 合成酶或该蛋白质的某些衍生物是与可溶性剪接活性有关。我们建议继续研究。具体目标是： (1) 确定准确的 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或共同发挥作用的多肽具有剪接活性。 (4) 确定 mt 酪氨酰-tRNA I组剪接中的合成酶和/或剪接活性功能内含子。 (5) 表征附加核基因和基因在脉孢菌中剪接 I 组内含子中发挥作用的产品线粒体。 (6) 在合作研究中，使用生化与针对脉孢菌开发的方法类似研究酵母线粒体中 I 组内含子的剪接，最初强调 mt 酪氨酰-tRNA 可能参与合成酶和其他 mt 氨酰基-tRNA 合成酶。这些研究旨在提供有关的基本信息催化活性RNA与所需蛋白质的相互作用催化活性并可以提供对进化的洞察真核生物中的内含子和剪接机制。