N⁶-methyladenosine in Mammalian Messenger RNA: Function, Location, and Quantitation

N-6-methyladenosine (m(6)A) is the most abundant internal modification in mammalian messenger RNA (mRNA), constituting 0.1 %-0.4 % of total adenosine residues in the transcriptome. m(6)A regulates mRNA stability and translation, pre-mRNA splicing, miRNA biogenesis, lncRNA binding, and many other physiological and pathological processes. While the majority of m(6)As occur in a consensus motif of DRm(6)ACH (D=A/G/U, R=A/G, H=U/A/C), the presence of such a motif does not guarantee methylation. Different RNA copies transcribed from the same gene may be methylated to varying levels. Within a single transcript, m(6)As are not evenly distributed, showing an enrichment in long internal and terminal exons. These characteristics of m(6)A deposition call for sequencing methods that not only pinpoint m6A sites at base resolution, but also quantitate the abundance of methylation across different RNA copies. In this review, we summarize existing m(6)A profiling methods, with an emphasis on next generation sequencing-(NGS-)based, site-specific, and quantitative methods, as well as several emerging single-cell methods.