Most m6A RNA Modifications in Protein-Coding Regions Are Evolutionarily Unconserved and Likely Nonfunctional.

Methylation of the adenosine base at the nitrogen-6 position (m6A) is the most prevalent internal posttranscriptional modification of mRNAs in many eukaryotes. Despite the rapid progress in the transcriptome-wide mapping of m6As, identification of proteins responsible for writing, reading, and erasing m6As, and elucidation of m6A functions in splicing, RNA stability, translation, and other processes, it is unknown whether most observed m6A modifications are functional. To address this question, we respectively analyze the evolutionary conservation of yeast and human m6As in protein-coding regions. Relative to comparable unmethylated As, m6As are overall no more conserved in yeasts and only slightly more conserved in mammals. Furthermore, yeast m6As and comparable unmethylated As have no significant difference in single nucleotide polymorphism (SNP) density or SNP site frequency spectrum. The same is true in human. The methylation status of a gene, not necessarily the specific sites methylated in the gene, is subject to purifying selection for no more than ∼20% of m6A-modified genes. These observations suggest that most m6A modifications in protein-coding regions are nonfunctional and nonadaptive, probably resulting from off-target activities of m6A methyltransferases. In addition, our reanalysis invalidates the recent claim of positive selection for newly acquired m6A modifications in human evolution. Regarding the small number of evolutionarily conserved m6As, evidence suggests that a large proportion of them are likely functional; they should be prioritized in future functional characterizations of m6As. Together, these findings have important implications for understanding the biological significance of m6A and other posttranscriptional modifications.