Abstract
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.
Highlights
Studies have estimated that alternative splicing can produce differently spliced messenger RNA transcripts for practically all multi-exon human genes [1,2]
Many of the alternative isoforms that we identified were only subtly different from the main splice variant, and one in five was generated by substitution of homologous exons by swapping one related exon for another
The alternative isoforms generated from homologous exons were highly conserved, first appearing 460 million years ago, and several appear to have tissue-specific roles in the brain and heart
Summary
Studies have estimated that alternative splicing can produce differently spliced messenger RNA (mRNA) transcripts for practically all multi-exon human genes [1,2]. These mRNA variants have the potential to expand the cellular protein repertoire far beyond the one gene–one protein model that formed part of the central dogma for many years [3,4]. The current version of the GENCODE gene set annotates more than 93,000 protein-coding variants, a number that has increased by 10,000 since 2009 All these transcripts could be translated into functional protein isoforms and could greatly diversify the cellular functional repertoire. All we can say is that most of the annotated splice variants in the human genome will produce isoforms with substantially altered 3D structure and consequent drastic change of biological function, if translated to protein [6,7]
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