ABSTRACT Recently, it was proposed that alternative splicing may act as a mechanism for opening

accelerated paths of evolution, by reducing negative selection pressure. What fraction of new gene features is

attributable to such a mechanism, and how can we assess whether they are biologically functional? To answer

these questions, we have analyzed metrics of very different types of evolutionary selection pressures (e.g. against

amino acid mutations (Ka/Ks); against mutations at synonymous sites (Ks); and for protein reading-frame

preservation: exons that are an exact multiple of 3nt in length can be spliced in or out without affecting the

downstream protein reading frame) to address this question via genome-wide analyses of human, chimpanzee,

mouse, and rat. These data show that alternative splicing relaxes Ka/Ks selection pressure up to seven-fold, but

intriguingly that this effect is accompanied by a strong increasein 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. Analysis of microarray

data for 3126 alternatively spliced exons across 10 mouse tissues generated by Pan and coworkers reveals that

frame-preserving exons are strongly associated with tissue-specific regulation of alternative splicing. Exons that

are alternatively spliced at uniformly high transcript inclusion levels or uniformly low levels show no preference for

protein frame-preservation. In contrast, alternatively spliced exons with dramatic changes of inclusion levels

across mouse tissues (referred to as “tissue-switched” exons) are both strikingly biased to be frame-preserving,

and are strongly conserved between human and mouse.