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.
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