Abstract
Alternative splicing is tightly regulated in a spatio-temporal and quantitative manner. This regulation is achieved by a complex interplay between spliceosomal (trans) factors that bind to different sequence (cis) elements. cis-elements reside in both introns and exons and may either enhance or silence splicing. Differential combinations of cis-elements allows for a huge diversity of overall splicing signals, together comprising a complex ‘splicing code’. Many cis-elements have been identified, and their effects on exon inclusion levels demonstrated in reporter systems. However, the impact of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs) on exon inclusion levels in human and chimpanzee. For this purpose, we compiled and studied comprehensive datasets of predicted ESRs, identified by several computational and experimental approaches, as well as microarray data for changes in alternative splicing levels between human and chimpanzee. Surprisingly, we found no association between changes in predicted ESRs and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5′ splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little or no effect on splicing, and thus interspecific changes at short-time scales may primarily occur in these effectively neutral ESRs. These results underscore the difficulties of using current computational ESR prediction algorithms to identify truly functionally important motifs, and provide a cautionary tale for studies of the effect of SNPs on splicing in human disease.
Highlights
Alternative splicing (AS) generates multiple transcripts from the same gene by differential splicing of introns, thereby increasing transcriptome and proteome diversity [1]
exonic splicing regulators (ESRs) density and change in alternatively spliced exons We studied ESR motif composition in 1845 alternatively spliced exons conserved between human and chimpanzee
A high fraction (57.4% to 87.5%, depending on the ESR dataset) of exonic nucleotides were part of at least one ESR hexamer, indicating that predicted ESRs are widely distributed across exons and that a very large proportion of exonic sequence might potentially impact splicing regulation (Table 1)
Summary
Alternative splicing (AS) generates multiple transcripts from the same gene by differential splicing of introns, thereby increasing transcriptome and proteome diversity [1]. Between 40–60% of all human genes [2,3,4,5] and up to 95% of multi-exon genes [6,7,8] are estimated to be alternatively spliced, and similar fractions have been estimated for other vertebrate species [9]. Multiple studies have shown that alternatively spliced exons are less conserved than constitutively spliced ones, suggesting that much alternative splicing may not be functional (reviewed in [12,13]). Expression of many alternatively spliced exons is highly regulated through development, including the precise regulation of exon inclusion levels (i.e. the fraction of transcripts from a given locus that include an exon; [14,15,16]). Several studies have shown that the precise regulation of AS is crucial for proper gene function Several studies have shown that the precise regulation of AS is crucial for proper gene function (e.g. [17,18,19]), evolutionary changes in AS regulation are likely to affect phenotype
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