Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level

Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64\% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20\% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved-all the homologous exons we identified evolved over 460 million years ago-and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles.This work was supported by the National Institutes of Health [grant number U41 HG007234], by the Spanish Ministry of Science and Innovation [grant numbers BIO2012-40205, BIO2012/37926 and RD07-0067-0014, COMBIOMED] and by the ``Instituto de Salud Carlos III´´ [grant numbers RIC-RD12/0042/0056 and PT13/0001/0017]. JMR was supported by the Spanish National Institute of Bioinformatics (www.inab.org), a platform of the ``Instituto de Salud Carlos III´´ [INB-ISCIII, PRB2]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S