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. 2008 Apr;18(4):533-43.
doi: 10.1101/gr.070268.107. Epub 2008 Jan 18.

Positive selection acting on splicing motifs reflects compensatory evolution

Shengdong Ke  1 , Xiang H-F Zhang, Lawrence A Chasin
Affiliations

Positive selection acting on splicing motifs reflects compensatory evolution

Shengdong Ke et al. Genome Res. 2008 Apr.

Abstract

We have used comparative genomics to characterize the evolutionary behavior of predicted splicing regulatory motifs. Using base substitution rates in intronic regions as a calibrator for neutral change, we found a strong avoidance of synonymous substitutions that disrupt predicted exonic splicing enhancers or create predicted exonic splicing silencers. These results attest to the functionality of the hexameric motif set used and suggest that they are subject to purifying selection. We also found that synonymous substitutions in constitutive exons tend to create exonic splicing enhancers and to disrupt exonic splicing silencers, implying positive selection for these splicing promoting events. We present evidence that this positive selection is the result of splicing-positive events compensating for splicing-negative events as well as for mutations that weaken splice-site sequences. Such compensatory events include nonsynonymous mutations, synonymous mutations, and mutations at splice sites. Compensation was also seen from the fact that orthologous exons tend to maintain the same number of predicted splicing motifs. Our data fit a splicing compensation model of exon evolution, in which selection for splicing-positive mutations takes place to counter the effect of an ongoing splicing-negative mutational process, with the exon as a whole being conserved as a unit of splicing. In the course of this analysis, we observed that synonymous positions in general are conserved relative to intronic sequences, suggesting that messenger RNA molecules are rich in sequence information for functions beyond protein coding and splicing.

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Figures

Figure 1.
Figure 1.
Rates of ESE and ESS creation and disruption. Rates have been normalized for change at intronic sites, expressed as Ks/Ki. Black bars represent ESE and ESS motifs (but purged of reverse complements); the gray bars represent two separate control motif sets, being largely the reverse complements of the ESE and ESS sets (see text). Error bars show 95% confidence intervals. (A,B) Human–macaque comparisons; (C,D) human–chimpanzee comparisons. (A,C) Constitutive exons; (B,D) alternatively spliced exons.
Figure 2.
Figure 2.
Distribution of Ks/Ki ratios for hexamers. Histograms of Ks/Ki values for ESEs, ESSs, and remaining hexamers are shown. Data include the full 469 ESE and 246 ESS sets (not purged of reverse complements) and 3448 non-ESXs and exclude changes at CpG sites and 33 mostly CpG-rich hexamers that yielded no data.
Figure 3.
Figure 3.
Evolutionary compensation of exonic splicing-negative motif changes by exonic splicing-positive motif changes. (A) Comparing human to macaque: exons were separated into four groups according to their frequency of exonic splicing-negative events (events per nt) as indicated. Exons with frequencies of 0–0.015, 0.015–0.030, and 0.030–0.045 generally contain 1 or 2, 3 or 4, and 5 or 6 splicing-negative differences, respectively. Exon pairs with frequencies higher than 0.045 have been omitted as many of them are poorly aligned. Black bars: real exons; white bars, simulated exons. (B) Net difference between the real and simulated sets is shown for each splicing-negative mutation category. (C) Same analysis as in A, except restricted to splicing changes in which the directionality of the change was determined by the use of dog as an out-group. Thus, in this panel the mutations in human exons can be considered ESE and ESS creations and disruptions rather than simply differences from macaque. (D) Same analysis as in A, but measuring differences using two randomly chosen non-overlapping sets of equal numbers of non-ESEs and of non-ESSs as controls. Similar results comparing macaque exons to human exons are presented in Supplemental Fig. S2.
Figure 4.
Figure 4.
Compensation between splice-site changes and exonic splicing motif changes (comparing human to macaque). (A) Splicing-positive splice-site changes correlate with splicing-negative motif changes and vice versa. The exonic splicing motif difference (ESMD) is defined as the frequency of splicing-positive changes (ESE creations and ESS disruptions) minus the frequency of splicing-negative changes (ESS creations and ESE disruptions) in human relative to macaque. A positive ESMD is predicted to promote splicing while a negative ESMD would discourage splicing. Exons in the “weaker” set have one splice site at which the CV score (consensus values) has decreased by at least 5 on a CV scale of 0–100 and in which the other splice site has not increased by >5. Exons in the “stronger” set are defined in the opposite way. Exons in the “unchanged” set show no change at all in CV score for both 3′ and 5′ splice sites. The results of a control using non-ESE/ESS motifs, as described in the legend to Fig. 3D, are shown on the right. (B) Proportion of exons that have undergone changes in splice-site sequence and splicing motifs reflects compensation. Standard errors are indicated in both panels. The total number of exons in the weaker, unchanged, and stronger sets are 2897, 20,855, and 3394, respectively. Similar results comparing macaque exons to human are presented in Supplemental Fig. S3.
Figure 5.
Figure 5.
Examples of compensatory and noncompensatory changes. Top sequences are human, bottom sequences macaque. Mutations are shaded, ESEs are underlined, ESSs are in bold italics, exons are in upper case, and introns are in lower case. (A) Decrease of an ESE in human compensated by the decrease of an ESS. Mutation 2 results in the absence of an ESE, potentially compensated by the presence of an ESS (mutation 1). (B) Weakened splice site compensated by an increase of an ESE or decrease of an ESS or both. Mutation 7 weakens the 5′ splice-site CV score from 89.4 to 77.4 (arrow), while mutations 1, 2, 3, and 4 result in ESE appearances and mutation 3 disrupts an ESS as well. Mutations 5 and 6 are predicted to be neutral. (C) Decrease in ESEs and increase in ESSs with no compensation. Mutations 1, 3, and 4 disrupt ESEs and mutation 2 creates two overlapping ESSs, with no further changes in the exon.
Figure 6.
Figure 6.
Global maintenance of exonic splicing motifs (comparing human to macaque). Motif change is defined as the number of ESE or ESS hexamers in a human exon minus the number in its macaque ortholog. Splicing promotion for an exon is defined as the ESE number minus the ESS number for that exon. In the simulated sets, the same number and type of differences seen in human exons were placed randomly among synonymous sites of macaque exons, as described in the text. Similar results simulating macaque differences in human exons are shown in Supplemental Fig. S4.
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