Sequence determinants and evolution of constitutive and alternative splicing in yeast species

Abstract

RNA splicing is a key process in eukaryotic gene expression. Most Intron-containing genes are constitutively spliced, hence efficient splicing of an intron is crucial for efficient gene expression. Here we use a large synthetic oligo library of ~20,000 variants to explore how different intronic sequence features affect splicing efficiency and mRNA expression levels in S. cerevisiae. Using a combinatorial design of synthetic introns we demonstrate how non-consensus splice site sequences affect splicing efficiency in each of the three splice sites. We then show that S. cerevisiae splicing machinery tends to select alternative 3 splice sites downstream of the original site, and we suggest that this tendency created a selective pressure, leading to the avoidance of cryptic splice site motifs near introns 3 ends. We further use natural intronic sequences from other yeast species, whose splicing machineries have diverged to various extents, to show how intron architectures in the various species have been adapted to the organisms splicing machinery. We suggest that the observed tendency for cryptic splicing is a result of a loss of a specific splicing factor, U2AF1. Lastly, we show that synthetic sequences containing two introns give rise to alternative RNA isoforms in S. cerevisiae, exposing intronic features that control and facilitate alternative splicing. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome.