Splicing in Drosophila

Alternative pre-mRNA splicing is a regulated step in eukaryotic gene expression in which introns are removed from the transcript and exons are joined together to form a mature mRNA. To study the effects of mutations on alternative splicing, we built a mutant library containing all combinations of 12 mutations that accumulated during the emergence of an alternatively-spliced human exon: FAS exon 6. This allowed us to study the effects of individual nucleotide substitutions in thousands of different closely-related genetic contexts. We show that the effect of the same mutation on exon inclusion depends non-monotonically on the inclusion levels of the exon before the mutation is made, with mutations having their strongest splice-altering effects in exons with intermediate levels of inclusion, and their smallest effects in exons that are always skipped or always included. After performing deep mutagenesis in two highly included (constitutive) exons, we find that, in agreement with our previous results, mutations have a very small effect on the inclusion of constitutive exons with the exception of mutations in exon-intron boundaries (splice sites). Since alternative splicing is frequently perturbed in human genetic diseases, we then put these results in a more practical context to address the question of the extent to which random mutations in the human genome are likely to have splice-altering effects. Since most human exons are highly included, we conclude that a random mutation is unlikely to have a splice altering effect, and that altered splicing should only be considered a likely proximal disease mechanism for mutations that affect alternatively spliced exons (included at intermediate levels) or disrupt splice sites.