Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements

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Abstract
Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs). Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5′ and 94% altered 3′ splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%–45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%–50% of ISREs were enriched near alternatively spliced (AS) exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events. During RNA splicing, sequences (introns) in a pre-mRNA are excised and discarded, and the remaining sequences (exons) are joined to form the mature RNA. Splicing is regulated not only by the binding of the basic splicing machinery to splice sites located at the exon–intron boundaries, but also by the combined effects of various other splicing factors that bind to a multitude of sequence elements located both in the exons as well as the flanking introns. Instances of alternative splicing, where usage of splice site(s) is incomplete or different between tissues, cell types, or lineages, can be created by the interaction of sequence elements and tissue, cell type, and stage-specific splicing factors. To better understand constitutive and alternative pre-mRNA splicing, the authors describe a comparative genomics approach, using available mammalian genomes, to systematically identify splicing regulatory elements located in the introns proximal to exons. A quarter of the elements were tested experimentally, and most of them altered splicing in human cells. The authors also showed that that the intronic elements are close to tissue-specific alternative exons and are more likely to be located in specific positions in the introns, suggestive of potential regulatory function. These elements are also frequently found in tissue-specific genes, suggesting a coupling between expression and alternative splicing of these genes. Finally, the authors propose a strategy using the elements to identify the binding sites of several splicing factors.