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

N⁶-methyladenosine (m⁶A) is the most abundant internal modification in mammalian messenger RNA (mRNA), constituting 0.1 %–0.4 % of total adenosine residues in the transcriptome. m⁶A regulates mRNA stability and translation, pre-mRNA splicing, miRNA biogenesis, lncRNA binding, and many other physiological and pathological processes. While the majority of m⁶As occur in a consensus motif of DRm⁶ACH (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, m⁶As are not evenly distributed, showing an enrichment in long internal and terminal exons. These characteristics of m⁶A 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 m⁶A profiling methods, with an emphasis on next generation sequencing-(NGS−)based, site-specific, and quantitative methods, as well as several emerging single-cell methods.