Accumulation of pre‐tRNA splicing ‘2/3’ intermediates in a Saccharomyces cerevisiae mutant.

In an effort to identify genes involved in the excision of tRNA introns in Saccharomyces cerevisiae, temperature‐sensitive mutants were screened for intracellular accumulation of intron‐containing tRNA precursors by RNA hybridization analysis. In one mutant, tRNA splicing intermediates consisting of the 5′ exon covalently joined to the intron (‘2/3’ pre‐tRNA molecules) were detected in addition to unspliced precursors. The mutant cleaves pre‐tRNA(Phe) in vitro at the 3′ exon/intron splice site, generating the 3′ half molecule and 2/3 intermediate. The 5′ half molecule and intron are not produced, indicating that cleavage at the 5′ splice site is suppressed. This partial splicing activity co‐purifies with tRNA endonuclease throughout several chromatographic steps. Surprisingly, the splicing defect does not appreciably affect cell growth at normal or elevated temperatures, but does confer a pseudo cold‐sensitive phenotype of retarded growth at 15 degrees C. The mutant falls into the complementation group SEN2 previously defined by the isolation of mutants defective for tRNA splicing in vitro [Winey, M. and Culbertson, M.R. (1988) Genetics, 118, 609‐617], although its phenotypes are distinct from those of the previous sen2 isolates. The distinguishing genetic and biochemical properties of this new allele, designated sen2‐3, suggests the direct participation of the SEN2 gene product in tRNA endonuclease function.