Transcript Specificity in Yeast Pre-mRNA Splicing Revealed by Mutations in Core Spliceosomal Components

Abstract

Appropriate expression of most eukaryotic genes requires the removal of introns from their pre–messenger RNAs (pre-mRNAs), a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well. The spliceosome is a large RNA-protein machine responsible for removing the noncoding (intron) sequences that interrupt eukaryotic genes. Nearly everything known about the behavior of this machine has been based on the analysis of only a handful of genes, despite the fact that individual introns vary greatly in both size and sequence. Here we have utilized a microarray-based platform that allows us to simultaneously examine the behavior of all intron-containing genes in the budding yeast S. cerevisiae. By systematically examining the effects of individual mutants in the spliceosome on the splicing of all substrates, we have uncovered a surprisingly complex relationship between the spliceosome and its full complement of substrates. Contrary to the idea that the spliceosome engages in “generic” interactions with all intron-containing substrates in the cell, our results show that the identity of the transcript can differentially affect splicing efficiency when the machine is subtly perturbed. We propose that the wild-type spliceosome can also distinguish among its many substrates as external conditions warrant to function as a specific regulator of gene expression.