Background: Alternative splicing, particularly through intron retention and exon skipping, is a major layer of pre-translational regulation in eukaryotes. While intron retention is believed to be the most prevalent mode across non-animal eukaryotes, animals have unusually high rates of exon skipping. However, when and how this high prevalence of exon skipping evolved is unknown. Since exon skipping can greatly expand proteomes, answering these questions sheds light on the evolution of higher organismal complexity in metazoans. Results: We used RNA-seq data to quantify exon skipping and intron retention frequencies across 65 eukaryotic species, with particular focus on early branching animals and unicellular holozoans. We found that only bilaterians have significantly increased their exon skipping frequencies compared to all other eukaryotic groups. Unlike in other eukaryotes, however, exon skipping in nearly all animals, including non-bilaterians, is strongly enriched for frame-preserving sequences, suggesting that exon skipping involvement in proteome expansion predated the increase in frequency. We also identified architectural features consistently associated with higher exon skipping rates within all studied eukaryotic genomes. Remarkably, these architectures became more prevalent during animal evolution, indicating co-evolution between genome architectures and exon skipping frequencies. Conclusion: We suggest that the increase of exon skipping rates in animals followed a two-step process. First, exon skipping in early animals became enriched for frame-preserving events. Second, bilaterian ancestors dramatically increased their exon skipping frequencies, likely driven by the interplay between a shift in their genome architectures towards more exon definition and recruitment of frame-preserving exon skipping events to functionally diversify their cell-specific proteomes.

This work was supported by the following grants to MI: European Research Council (ERC) Starting Grant (agreement ERC-StG-LS2–637591, under the EU Horizon 2020 Plan) and the Spanish Ministry of Economy and Competitiveness (MINECO, agreement BFU2014–55076-P; part of the Severo Ochoa Excellence Centre plan 2013–2017, SEV-2012-0208). It was also supported by the following research grants to IRT: ERC Consolidator (ERC-2012-Co-616960), the Secretary’s Office for Universities and Research of the Generalitat de Catalunya (2014 SGR 619) and MINECO (MINECO BFU2014–57779-P, with European Regional Development Fund support). XGB was supported by a pre-doctoral FPI grant from MINECO and the ERC Consolidator Grant to IRT.