Genome-wide analyses of alternative splicing have established its nearly ubiquitous role in gene regulation in many organisms. Genome sequencing and comparative genomics have made it possible to look in detail at the evolutionary history of specific alternative exons or splice sites, resulting in a flurry of publications in recent years. Here, we consider how alternative splicing has contributed to the evolution of modern genomes, and discuss constraints on evolution associated with alternative splicing that might have important medical implications.

Recently, it was proposed that alternative splicing may act as a mechanism for opening accelerated paths of evolution, by reducing negative selection pressure, but there has been little evidence so far that this mechanism could produce adaptive benefit. Here, we use metrics of very different types of selection pressures [e.g., against amino acid mutations (Ka/Ks), against mutations at synonymous sites (Ks), and for protein reading-frame preservation] to address this question by genomewide analyses of human, chimpanzee, mouse, and rat. These data show that alternative splicing relaxes Ka/Ks selection pressure up to 7-fold, but intriguingly this effect is accompanied by a strong increase in selection pressure against synonymous mutations, which propagates into the adjacent intron, and correlates strongly with the alternative splicing level observed for each exon. These effects are highly local to the alternatively spliced exon. Comparisons of these four genomes consistently show an increase in the density of amino acid mutations (Ka) in alternatively spliced exons and a decrease in the density of synonymous mutations (Ks). This selection pressure against synonymous mutations in alternatively spliced exons was accompanied in all four genomes by a striking increase in selection pressure for protein reading-frame preservation, and both increased markedly with increasing evolutionary age. Restricting our analysis to a subset of exons with strong evidence for biologically functional alternative splicing produced identical results. Thus alternative splicing apparently can create evolutionary “hotspots” within a protein sequence, and these events have evidently been selected for during mammalian evolution.

Recent genome-wide analyses of alternative splicing indicate that 40−60% of human genes have alternative splice forms, suggesting that alternative splicing is one of the most significant components of the functional complexity of the human genome. Here we review these recent results from bioinformatics studies, assess their reliability and consider the impact of alternative splicing on biological functions. Although the 'big picture' of alternative splicing that is emerging from genomics is exciting, there are many challenges. High-throughput experimental verification of alternative splice forms, functional characterization, and regulation of alternative splicing are key directions for research. We recommend a community-based effort to discover and characterize alternative splice forms comprehensively throughout the human genome.

Single nucleotide polymorphisms in protein coding regions (cSNPs) are of great interest for their effects on phenotype and potential for mapping disease genes. We have identified 5400 novel exonic SNPs from alignments of public EST data to the draft human genome sequence, and approximately 12 000 more novel exonic SNPs from EST cluster alignments. We found 82% of the genomic-aligned SNPs and 63% of the EST-only SNPs to be detectably polymorphic in 20 Finnish DNA samples. 37% of the SNPs mapped to known protein coding regions, yielding 6500 distinct, novel cSNPs from the two datasets. These data reveal selection against mutations that alter protein structure, and distinct classes of genes under strongly positive vs. negative pressure from natural selection for amino acid replacement (detected by KA/KSratio). We have searched these cSNPs for compatibility with the amino acid profile at each site and structural impact on protein core stability.

Single-nucleotide polymorphisms (SNPs) have been explored as a high-resolution marker set for accelerating the mapping of disease genes1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. Here we report 48,196 candidate SNPs detected by statistical analysis of human expressed sequence tags (ESTs), associated primarily with coding regions of genes. We used Bayesian inference to weigh evidence for true polymorphism versus sequencing error, misalignment or ambiguity, misclustering or chimaeric EST sequences, assessing data such as raw chromatogram height, sharpness, overlap and spacing, sequencing error rates, context-sensitivity and cDNA library origin. Three separate validations—comparison with 54 genes screened for SNPs independently, verification of HLA-A polymorphisms and restriction fragment length polymorphism (RFLP) testing—verified 70%, 89% and 71% of our predicted SNPs, respectively. Our method detects tenfold more true HLA-A SNPs than previous analyses of the EST data. We found SNPs in a large fraction of known disease genes, including some disease-causing mutations (for example, the HbS sickle-cell mutation). Our comprehensive analysis of human coding region polymorphism provides a public resource for mapping of disease genes (available at http://www.bioinformatics.ucla.edu/snp).