Document Type



Life Sciences


The proteome expanding effects of alternative pre-mRNA splicing have had a profound impact on eukaryotic evolution. The events that create this diversity can be placed into four major classes: exon skipping, intron retention, alternative 5′ splice sites, and alternative 3′ splice sites. Although the regulatory mechanisms and evolutionary pressures among alternative splicing classes clearly differ, how these differences affect the evolution of splicing regulation remains poorly characterized. We used RNA-seq to investigate splicing differences in D. simulans, D. sechellia, and three strains of D. melanogaster. Regulation of exon skipping and tandem alternative 3′ splice sites (NAGNAGs) were more divergent than other splicing classes. Splicing regulation was most divergent in frame-preserving events and events in noncoding regions. We further determined the contributions of cis- and trans-acting changes in splicing regulatory networks by comparing allele-specific splicing in F1 interspecific hybrids, because differences in allele-specific splicing reflect changes in cis-regulatory element activity. We find that species-specific differences in intron retention and alternative splice site usage are primarily attributable to changes in cis-regulatory elements (median ∼80% cis), whereas species-specific exon skipping differences are driven by both cis- and trans-regulatory divergence (median ∼50% cis). These results help define the mechanisms and constraints that influence splicing regulatory evolution and show that networks regulating the four major classes of alternative splicing diverge through different genetic mechanisms. We propose a model in which differences in regulatory network architecture among classes of alternative splicing affect the evolution of splicing regulation.


Originally published in :

Genome Res. May 2014; 24(5): 786–796. doi: 10.1101/gr.161521.113 PMID:24515119 PMCID: PMC4009608

Data access

High-throughput sequencing data have been submitted to the NCBI Sequence Read Archive (SRA; under accession number SRP023274. Genome sequences are accessible at the McManus (, Wittkopp (∼pwlab/), and Graveley ( laboratory websites. Contigs with novel sequences from D. simulans and D. sechellia have been submitted to GenBank ( under accession numbers JAQJ00000000 and JAQR00000000, respectively.

Included in

Life Sciences Commons