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

Methylation of the adenosine base at the nitrogen-6 position (m(6)A) is the most prevalent internal posttranscriptional modification of mRNAs in many eukaryotes. Despite the rapid progress in the transcriptome-wide mapping of m(6)As, identification of proteins responsible for writing, reading, and erasing m(6)As, and elucidation of m(6)A functions in splicing, RNA stability, translation, and other processes, it is unknown whether most observed m(6)A modifications are functional. To address this question, we respectively analyze the evolutionary conservation of yeast and human m(6)As in protein-coding regions. Relative to comparable unmethylated As, m(6)As are overall no more conserved in yeasts and only slightly more conserved in mammals. Furthermore, yeast m(6)As 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 similar to 20% of m(6)A-modified genes. These observations suggest that most m(6)A modifications in protein-coding regions are nonfunctional and nonadaptive, probably resulting from off-target activities of m(6)A methyltransferases. In addition, our reanalysis invalidates the recent claim of positive selection for newly acquired m(6)A modifications in human evolution. Regarding the small number of evolutionarily conserved m(6)As, evidence suggests that a large proportion of them are likely functional; they should be prioritized in future functional characterizations of m(6)As. Together, these findings have important implications for understanding the biological significance of m(6)A and other posttranscriptional modifications.