Neutral substitutions occur at a faster rate in exons than in noncoding DNA in primate genomes

Abstract

Point mutation rates in exons (synonymous sites) and noncoding (introns and intergenic) regions are generally assumed to be the same. However, comparative sequence analyses of synonymous substitutions in exons (81 genes) and that of long intergenic fragments (141.3 kbp) of human and chimpanzee genomes reveal a 30%-60% higher mutation rate in exons than in noncoding DNA. We propose a differential CpG content hypothesis to explain this fundamental, and seemingly unintuitive, pattern. We find that the increased exonic rate is the result of the relative overabundance of synonymous sites involved in CpG dinucleotides, as the evolutionary divergence in non-CpG sites is similar in noncoding DNA and synonymous sites of exons. Expectations and predictions of our hypothesis are confirmed in comparisons involving more distantly related species, including human-orangutan, human-baboon, and human-macaque. Our results suggest an underlying mechanism for higher mutation rate in GC-rich genomic regions, predict nonlinear accumulation of mutations in pseudogenes over time, and provide a possible explanation for the observed higher diversity of single nucleotide polymorphisms (SNPs) in the synonymous sites of exons compared to the noncoding regions.

Figure 1.

(A) Evolutionary divergence per 100 sites and (B) CpG contents of synonymous sites in exons, pseudogenes, introns, and intergenic DNA for the human–chimpanzee comparison. The mean and the standard error estimates shown were obtained from 81 protein coding genes, 19 pseudogenes, introns of 48 genes, and four intergenic blocks. In (A), the total height of each column (including black and open bars) corresponds to the overall evolutionary divergence. The black portion in each column depicts divergence with CpG sites excluded, and therefore, the open bars show the contribution of mutations at CpG sites to the overall divergence. In (B), the CpG content is the proportion of sites involved in the CpG dinucleotide configuration. For noncoding DNA, all sites were included for the estimation of CpG content and evolutionary divergence, whereas for exons only the fourfold-degenerate sites were included (see Methods).

Figure 2.

Relationship between evolutionary divergence and the ratio of CpG contents in pseudogenes and their functional counterparts. (A) Analysis of 39 human pseudogenes [R² = 0.52; P < 0.01], (B) seven pseudogenes created from the same Arginino succinate synthetase gene [R² = 0.90; P < 0.01], and (C) five pseudogenes of the same Beta tubulin Q gene [R² = 0.73; P < 0.05]. The relative CpG content is the ratio of the overall CpG content in the pseudogene divided by overall CpG content of the functional gene.

Figure 3.

(A) Relationship between evolutionary divergence and GC content, GC₄, at synonymous sites of 93 genes from the human–macaque comparison; R² = 0.125, P < 0.01. (B) Relationship between evolutionary divergence and GC₄ after excluding fourfold-degenerate sites involved in CpG dinucleotides; R² = 0.01, P > 0.05.

Figure 4.

(A) Relationship between synonymous divergence in exons and evolutionary divergence in introns; R² = 0.511, P < 0.01. (B) Relationship between synonymous divergence in exons and evolutionary divergence in introns after removing CpG sites; R² = 0.137, P > 0.05. With the rightmost outlier excluded, R² = 0.06, P > 0.05.