Sequence determinants of splicing in yeast

mRNA splicing is a key process in eukaryotic gene expression. Most Intron-containing genes are constitutively spliced, and hence must undergo splicing to produce a functional mRNA. Therefore, regulation of splicing efficiency greatly affects gene expression regulation. Here we use a large synthetic oligo library of ~25,000 variants to explore how different intronic sequence determinants affect splicing efficiency and mRNA expression levels in S. cerevisiae. We found that the three splice sites (donor, acceptor, and branching point) differ in how deviations from the consensus sequence affect functionality. We also use intronic sequences from other yeast species with modified splicing machinery to show that intron architecture has co-evolved with the splicing machinery to adapt to the presence or absence of a specific splicing factor. Finally, we show that synthetic sequences containing two introns give rise to diverse RNA isoforms, 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.