mRNA splicing is one of the key processes in eukaryotic gene expression. Most Intron-containing genes are constitutively spliced, and hence must undergo splicing in order to produce a functional mature mRNA. Therefore, regulation of splicing efficiency greatly affects broader 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 yeast. 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, which enables us to elucidate intronic features that control and enable alternative splicing. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome.