Mutations in m6A consensus motifs are suppressed in the m6A modified genes in human cancer cells

N-6-methyladenosine (m(6)A) is the most prevalent type of RNA modification. METTL3 in the methyltransferase complex is the core enzyme responsible for methylation. METTL3 selectively catalyzes the adenosines centered in the RRAC motif. Functional studies established that m(6)A could enhance the translation efficiency (TE) of modified genes by recruiting reader protein YTHDF1 and other initiation factors. We downloaded the m(6)A peaks in HeLa cells from a previous study and defined the m(6)A modified genes and sites. Ancestral mutations in the genic region fixed in the HeLa cell samples were defined using their mRNA-Seq data and the alignment between human and mouse genomes. Furthermore, in the small interfering (si)-METTL3sample, the calculated TE foldchange of all genes was compared to that in the negative control. The TE of m(6)A genes was globally down-regulated in si-METTL3versus control compared to the non-m(6)A genes. In m(6)A modified genes, RRAC motif mutations were suppressed compared to mutations in non-motif regions or non-m(6)A genes. Among the m(6)A genes, a fraction RRAC motif mutations negatively correlated with the TE foldchange (si-METTL3versus control). The TE of m(6)A modified genes was enhanced in HeLa cells. RRAC motif mutations could potentially prevent methylation of adenosines and consequently abolish the enhanced translation. Such mutations in the RRAC motif might be deleterious. Accordingly, we observed lower fractions of mutations in RRAC motifs than in other regions. This prevention of mutations in the RRAC motif could be a strategy adopted by cancer cells to maintain the elevated translation of particular genes.