A yeast tRNA precursor containing a pre-mRNA intron is spliced via the pre-mRNA splicing mechanism

We have replaced the 14 nucleotide long intervening sequence of the Saccharomyces cerevisiae SUP6 (ochre) tRNA(Tyr) gene by the 52 nucleotide long second intron of the S. cerevisiae MATa1 gene. Yeast cells containing this modified pre-tRNA showed the typical suppressor phenotype indicating that the MATa1 pre-mRNA intron was exactly excised in vivo from the primary tRNA transcript and a mature and functional tRNA was formed. Several lines of evidence show that the splicing reaction proceeded via the pre-mRNA splicing mechanism: the reaction yielded a lariat shaped excised intron with a lariat shaped intron-exon 2 molecule as intermediate; point mutations in the conserved UAC-UAAC box of the intron impaired splicing of the precursor RNA; in a temperature sensitive rna2 strain splicing of this tRNA precursor was inhibited at the restrictive temperature. Our results imply that in yeast the excision of a pre-mRNA intron is not dependent on the transcription apparatus by which it was generated and that transcription and splicing are uncoupled processes in vivo, too. Furthermore these data demonstrate that recognition of an RNA as a substrate for a pre-mRNA splicing reaction is, at least qualitatively, only intron dependent.