Although the assumption of the neutral theory of molecular evolution - that some classes of mutation have too small an effect on fitness to be affected by natural selection - seems intuitively reasonable, over the past few decades the theory has been in retreat. At least in species with large populations, even synonymous mutations in exons are not neutral. By contrast, in mammals, neutrality of these mutations is still commonly assumed. However, new evidence indicates that even some synonymous mutations are subject to constraint, often because they affect splicing and/or mRNA stability. This has implications for understanding disease, optimizing transgene design, detecting positive selection and estimating the mutation rate.

Silent sites in mammals have classically been assumed to be free from selective pressures. Consequently, the synonymous substitution rate (Ks) is often used as a proxy for the mutation rate. Although accumulating evidence demonstrates that the assumption is not valid, the mechanism by which selection acts remain unclear. Recent work has revealed that the presence of exonic splicing enhancers (ESEs) in coding sequence might influence synonymous evolution. ESEs are predominantly located near intron-exon junctions, which may explain the reduced single-nucleotide polymorphism (SNP) density in these regions. Here we show that synonymous sites in putative ESEs evolve more slowly than the remaining exonic sequence. Differential mutabilities of ESEs do not appear to explain this difference. We observe that substitution frequency at fourfold synonymous sites decreases as one approaches the ends of exons, consistent with the existing SNP data. This gradient is at least in part explained by ESEs being more abundant near junctions. Between-gene variation in Ks is hence partly explained by the proportion of the gene that acts as an ESE. Given the relative abundance of ESEs and the reduced rates of synonymous divergence within them, we estimate that constraints on synonymous evolution within ESEs causes the true mutation rate to be underestimated by not more than approximately 8%. We also find that Ks outside of ESEs is much lower in alternatively spliced exons than in constitutive exons, implying that other causes of selection on synonymous mutations exist. Additionally, selection on ESEs appears to affect nonsynonymous sites and may explain why amino acid usage near intron-exon junctions is nonrandom.

Simulating evolution and reallocating the substitutions observed in mouse genes revealed that in mammals synonymous sites do not evolve neutrally and synonymous mutations may be under selection because of their effects on the thermodynamic stability of mRNA.

In mammals divergence at fourfold degenerate sites in codons (K(4)) and intronic sequence (K(i)) are both used to estimate the mutation rate, under the supposition that both evolve neutrally. Does it matter which of these we use? Using either class of sequence can be defended because (1) K(4) is the same as K(i) (at least in rodents) and (2) there is no selectively driven codon usage (hence no systematic selection on third sites). Here we re-examine these findings using 560 introns (for 136 genes) in the mouse-rat comparison, aligned by eye and using a new maximum likelihood protocol. We find that the rate of evolution at fourfold sites and at intronic sites is similar in magnitude, but only after eliminating putatively constrained sites from introns (first introns and sites flanking intron-exon junctions). Any approximate congruence between the two rates is not, however, owing to an underlying similarity in the mode of sequence evolution. Some dinucleotides are hypermutable and differently abundant in exons and introns (e.g., CpGs). More importantly, after controlling for relative abundance, all dinucleotides starting with A or T are more prevalent in mismatches in exons than in introns, whereas C-starting dinucleotides (except CG) are more common in introns. Although C content at intronic sites is lower than at flanking fourfold sites, G content is similar, demonstrating that there exists a strong strand-specific preference for C nucleotides that is unique to exons. Transcription-coupled mutational processes and biased gene conversion cannot explain this, as they should affect introns and flanking exons equally. Therefore, by elimination, we propose this to be strong evidence for selectively driven codon usage in mammals.

No abstract