In Vivo Pre-tRNA Processing in Saccharomyces cerevisiae

Abstract

We have surveyed intron-containing RNAs of the yeast Saccharomyces cerevisiae by filter hybridization with pre-tRNA intron-specific oligonucleotide probes. We have classified various RNAs as pre-tRNAs, splicing intermediates, or excised intron products according to apparent size and structure. Linear, excised intron products were detected, and one example was isolated and sequenced directly. Additional probes designed to detect other precursor sequences were used to verify the identification of several intermediates. Pre-tRNA species with both 5′ leader and 3′ extension, with 3′ extension only, and with mature ends were distinguished. From these results, we conclude that the processing reactions used to remove the 5′ leader and 3′ extension from the transcript are ordered 5′ end trimming before 3′ end trimming. Splicing intermediates containing the 5′ exon plus the intron were detected. The splice site cleavage reactions are probably ordered 3′ splice site cleavage before 5′ splice site cleavage. Surprisingly, we also detected a splicing intermediate with the 5′ leader and a spliced product with both 5′ leader and 3′ extension. Evidently, splicing and end trimming are not ordered relative to each other, splicing occurring either before or after end trimming.