Human C-to-U Coding RNA Editing Is Largely Nonadaptive

Abstract

C-to-U RNA editing enzymatically converts the base C to U in RNA molecules and could lead to nonsynonymous changes when occurring in coding regions. Hundreds to thousands of coding sites were recently found to be C-to-U edited or editable in humans, but the biological significance of this phenomenon is elusive. Here, we test the prevailing hypothesis that nonsynonymous editing is beneficial because it provides a means for tissue- or time-specific regulation of protein function that may be hard to accomplish by mutations due to pleiotropy. The adaptive hypothesis predicts that the fraction of sites edited and the median proportion of RNA molecules edited (i.e., editing level) are both higher for nonsynonymous than synonymous editing. However, our empirical observations are opposite to these predictions. Furthermore, the frequency of nonsynonymous editing, relative to that of synonymous editing, declines as genes become functionally more important or evolutionarily more constrained, and the nonsynonymous editing level at a site is negatively correlated with the evolutionary conservation of the site. Together, these findings refute the adaptive hypothesis; they instead indicate that the reported C-to-U coding RNA editing is mostly slightly deleterious or neutral, probably resulting from off-target activities of editing enzymes. Along with similar conclusions on the more prevalent A-to-I editing and m6A modification of coding RNAs, our study suggests that, at least in humans, most events of each type of posttranscriptional coding RNA modification likely manifest cellular errors rather than adaptations, demanding a paradigm shift in the research of posttranscriptional modification.

Fig. 1.

Frequencies and editing levels of synonymous and nonsynonymous C-to-U RNA editing in human coding sequences. (A) Frequency of nonsynonymous editing is lower than that of synonymous editing in macrophages, monocytes, and HEK293T cells. P values are from χ² tests. (B) Nonsynonymous editing levels are lower than synonymous editing levels. In the boxplot, the lower edge and upper edge of a box represent the 25% quartile (q₁) and 75% quartile (q₃), respectively. The horizontal line inside a box indicates the median (md). The whiskers extend to the most extreme values inside inner fences, md ± 1.5(q₃−q₁). P values are from two-tailed Wilcoxon rank sum tests.

Fig. 2.

Nonsynonymous C-to-U RNA editing frequency (f_n), relative to synonymous editing frequency (f_s), reduces with gene importance and evolutionary constraint on the gene. (A) f_n/f_s is lower in essential genes than in nonessential genes. Open bars show the frequency distribution of f_n/f_s from 1,000 random sets of nonessential genes with the same number of genes as essential genes. The black arrow indicates the observed f_n/f_s in essential genes. (B) f_n/f_s increases with d_N/d_S, which is computed using human–mouse orthologs. We divided human genes into ten equal-sized bins by d_N/d_S, and each dot shows the f_n/f_s and median d_N/d_S of each bin. Spearman’s correlation coefficient (ρ) and its associated P value are shown.

Fig. 3.

Relationship between editing and evolutionary conservation. (A) Codons with a synonymously edited C and those with an unedited C that is potentially synonymously editable have similar evolutionary conservations at the amino acid level. (B) Codons with a nonsynonymously edited C and those with an unedited C that is potentially nonsynonymously editable have similar evolutionary conservations at the amino acid level. In the boxplot of (A) and (B), the lower edge and upper edge of a box represent the 25% quartile (q₁) and 75% quartile (q₃), respectively. The horizontal line inside a box indicates the median (md). The whiskers extend to the most extreme values inside inner fences, md ± 1.5(q₃ − q₁). P values are from Wilcoxon rank sum tests. (C) Synonymous editing level at a site is not significantly correlated with the evolutionary conservation (at the amino acid level) of the codon encompassing the site. Each dot is an edited site. (D) Nonsynonymous editing level at a site is significantly negatively correlated with the evolutionary conservation (at the amino acid level) of the codon encompassing the site. In (C) and (D), Spearman’s correlation coefficient (ρ) and its associated P value are shown.