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.
Highlights
Considering that the human genome contains upwards of 20,000 genes with an average of eight to ten exons per gene, it is remarkable that the RNA splicing machinery faithfully distinguishes exons from intronic sequences that are 100- to 1,000-fold larger in size
It reveals a set of evolutionarily conserved intronic splicing regulatory element (ISRE), and hundreds of candidate ISRE-regulated exons conserved across mammalian genomes
84% and 94% of tested ISREs were shown to suppress intron-proximal 59ss and 39ss in competing splice site reporter constructs in human cells, demonstrating that most ISREs can affect splicing
Summary
Considering that the human genome contains upwards of 20,000 genes with an average of eight to ten exons per gene, it is remarkable that the RNA splicing machinery faithfully distinguishes exons from intronic sequences that are 100- to 1,000-fold larger in size. Many studies show that the fidelity of splicing relies on cooperative interaction between the splicesomal complex and splicing trans factors (reviewed in [1]). Simple models of exon recognition depict trans factors binding to splicing regulatory elements (SREs) in cis that are in intronic regions proximal to the exon, or within the exon itself, resulting in either increased exon usage (splicing enhancers), or decreased splice site recognition (splicing silencers) [2,3,4]. In addition to regulating constitutive splicing (e.g., where a gene has only one isoform), SREs are important in regulating tissue-specific and developmentally regulated alternative splicing events [2,3,4]. Evidence that as many as 75% of human genes undergo alternative splicing, whereby multiple isoforms are derived from the same genic location, underscores the complexity of RNA splicing regulation [5]. A complete catalog of SREs is necessary to improve our understanding of the mechanisms controlling splicing, for it will enable rapid diagnosis and treatment of splicing-associated diseases [7]
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