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Intron Mutations Affect Splicing of Saccharomyces cerevisiae SUP53 Precursor tRNA

Strobel, Marjorie C. and Abelson, John (1986) Intron Mutations Affect Splicing of Saccharomyces cerevisiae SUP53 Precursor tRNA. Molecular and Cellular Biology, 6 (7). pp. 2674-2683. ISSN 0270-7306. PMCID PMC367824. doi:10.1128/MCB.6.7.2674.

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The Saccharomyces cerevisiae amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the role of intron structure and sequence on precursor tRNA splicing in vivo and in vitro. This gene encodes a pre-tRNA which contains a 32-base intervening sequence. Two types of SUP53 intron mutants were constructed: ones with an internal deletion of the natural SUP53 intron and ones with a novel intron. These mutant genes were transcribed in vitro, and the end-processed transcripts were analyzed for their ability to serve as substrates for the partially purified S. cerevisiae tRNA endonuclease and ligase. The in vitro phenotype of these mutant RNAs was correlated with the in vivo suppressor tRNA function of these SUP53 alleles after integration of the genes into the yeast genome. Analysis of these mutant pre-tRNAs, which exhibited no perturbation of the mature domain, clearly showed that intron structure and sequence can have profound effects on pre-tRNA splicing. All of the mutant RNAs, which were inefficiently spliced or unspliced, evidenced cleavage only at the 5' splice junction. Base changes in the intron proximal to the 3' splice junction could partially rescue the splicing defect. The implications of these data for tRNA endonuclease-substrate interactions are discussed.

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Additional Information:© 1986 American Society for Microbiology. Received 4 December 1985. Accepted 14 April 1986. We thank Eric Phizicky, Chris Greer, Andy Newman, and Rich Schwartz for their input and insights during the course of this work and Gayle Knapp for many stimulating discussions. Special thanks to Linda Brubaker, Connie Katz, and Donna Walker for preparation of the manuscript. Computer resources were provided by BIONET National Computer Research for Molecular Biology, whose funding is provided by the Biomedical Research Technology Program, Division of Research Resources, National Institutes of Health (grant 1-U41-RR-01685-02). This work was supported by the American Cancer Society (grant ACS NP302D) and by Public Health Service grant GM-32637 from the National Institutes of Health to J .A. M.C.S. was supported by a Public Health Service Genetics Training Grant (Biology Department, University of California, San Diego).
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American Cancer SocietyACS NP302D
Issue or Number:7
PubMed Central ID:PMC367824
Record Number:CaltechAUTHORS:20120627-140653187
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Official Citation:Intron mutations affect splicing of Saccharomyces cerevisiae SUP53 precursor tRNA. M C Strobel and J Abelson Mol. Cell. Biol. July 1986 6:2674-2683; doi:10.1128/MCB.6.7.2674
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:32153
Deposited By: Ruth Sustaita
Deposited On:27 Jun 2012 21:21
Last Modified:09 Nov 2021 21:25

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