Selection at silent sites
Chamary, J.V., Parmley, J.L. and Hurst, L.D. (2006) Hearing silence: non-neutral evolution at synonymous sites in mammals. Nature Reviews Genetics 7: 98-108
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.
Copyright 2006 Nature Publishing Group | PubMed entry | Request reprint
Parmley, J.L., Chamary, J.V. and Hurst, L.D. (2006) Evidence for purifying selection against synonymous mutations in mammalian exonic splicing enhancers. Molecular Biology and Evolution 23: 301-309
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(s) 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 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 four-fold 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 non-synonymous sites and may explain why amino acid usage near intron-exon junctions is non-random.
Copyright 2005 SMBE | PDF reprint of paper | PubMed entry | Supplementary data | Top of page
Chamary, J.V. and Hurst, L.D. (2005) Evidence for selection on synonymous mutations affecting stability of mRNA secondary structure in mammals. Genome Biology 6: R75
BACKGROUND: In mammals, contrary to what is usually assumed, recent evidence suggests that synonymous mutations may not be selectively neutral. This position has proven contentious, not least because of the absence of a viable mechanism. Here we test whether synonymous mutations might be under selection owing to their effects on the thermodynamic stability of mRNA, mediated by changes in secondary structure. RESULTS: We provide numerous lines of evidence that are all consistent with the above hypothesis. Most notably, by simulating evolution and reallocating the substitutions observed in the mouse lineage, we show that the location of synonymous mutations is non-random with respect to stability. Importantly, the preference for cytosine at 4-fold degenerate sites, diagnostic of selection, can be explained by its effect on mRNA stability. Likewise, by interchanging synonymous codons, we find naturally occurring mRNAs to be more sul than simulant transcripts. Housekeeping genes, whose proteins are under strong purifying selection, are also under the greatest pressure to maintain stability. CONCLUSION: Taken together, our results provide evidence that, in mammals, synonymous sites do not evolve neutrally, at least in part owing to selection on mRNA stability. This has implications for the application of synonymous divergence in estimating the mutation rate.
Copyright 2005 licensed by BioMed Central Ltd | PDF reprint of paper | PubMed entry | Supplementary data
Chamary, J.V. and Hurst, L.D. (2005) Biased codon usage near intron-exon junctions: selection on splicing enhancers, splice-site recognition or something else? Trends in Genetics 21: 256-259
Two groups recently argued that, in human genes, synonymous sites near intron-exon junctions are under selection for correct splicing. However, neither study controlled for the possibility of an underlying nucleotide bias at exon ends. We show that generalised A+T enrichment exists that could be independent of splicing regulation. Controlling for this bias, there remains evidence for selection between synonymous codons associated with splicing enhancers while support for cryptic splice site avoidance is diminished.
Copyright 2005 Elsevier Ltd | PDF reprint of paper | PubMed entry | Supplementary data | Top of page
Lercher, M.J., Chamary, J.V. and Hurst, L.D. (2004) Genomic regionality in rates of evolution is not explained by clustering of genes of comparable expression profile. Genome Research 14: 1002-1013
In mammalian genomes, linked genes show similar rates of evolution, both at fourfold degenerate synonymous sites (K4) and at nonsynonymous sites (KA). Although it has been suggested that the local similarity in the synonymous substitution rate is an artifact caused by the inclusion of disparately evolving gene pairs, we demonstrate here that this is not the case: after removal of disparately evolving genes, both (1) linked genes and (2) introns from the same gene have more similar silent substitution rates than expected by chance. What causes the local similarity in both synonymous and nonsynonymous substitution rates? One class of hypotheses argues that both may be related to the observed clustering of genes of comparable expression profile. We investigate these hypotheses using substitution rates from both human-mouse and mouse-rat comparisons, and employing three different methods to assay expression parameters. Although we confirm a negative correlation of expression breadth with both K4 and KA, we find no evidence that clustering of similarly expressed genes explains the clustering of genes of comparable substitution rates. If gene expression is not responsible, what about other causes? At least in the human-mouse comparison, the local similarity in KA can be explained by the covariation of KA and K4. As regards K4, our results appear consistent with the notion that local similarity is due to processes associated with meiotic recombination.
Copyright 2004 Cold Spring Harbor Lab. Press | PDF reprint of paper | PubMed entry | Supplementary data
Chamary, J.V. and Hurst, L.D. (2004) Similar rates but different modes of sequence evolution in introns and at exonic silent sites in rodents: evidence for selectively-driven codon usage. Molecular Biology and Evolution 21: 1014-1023
In mammals divergence at four-fold degenerate sites in codons (K4) and intronic sequence (Ki) 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) K4 is the same as Ki (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 four-fold 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 hypermuul 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, while C-starting dinucleotides (except CG) are more common in introns. While C content at intronic sites is lower than at flanking four-fold 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.