Figure 2. Deposition and detection of m⁶A.

(A) m⁶A is a low stoichiometry modification. At any given m⁶A site in a specific mRNA, only a small number (usually less than 20%) of the transcript copies in the cell will have m⁶A at that site. Most of the DRACH m⁶A consensus sites are not methylated (green circle). Additionally, some DRACH sites are more methylated than others, and all DRACH sites are likely methylated to some degree, although the stoichiometry is likely very low. The molecular mechanism that causes some DRACH sites to be more methylated than others, even in the same transcript, are not fully understood. Although m⁶A mapping may reveal multiple m⁶A sites for a given mRNA, the individual transcripts that comprise the m⁶A annotation are typically methylated only at a subset of sites. Newer single-transcript analysis methods have revealed the stochastic nature of methylation on individual transcripts.

(B) Gene architecture influences m⁶A deposition. Shown (left) are examples of two genes, one containing a long internal exon. After transcription (right), m⁶A is preferentially formed on the regions of transcripts corresponding to long exons. m⁶A is also enriched near the terminal exon-exon junction, particularly in mRNAs with long 3’UTRs. The correlation of m⁶A with these genomic features suggests that the writer complex is regulated by other events that are responsive to gene architecture.

(C) Putative differences in m⁶A are sometimes artifacts of the methods used to call m⁶A sites from mapping data. Many studies have used mapping methods such as MeRIP-seq to map m⁶A and to compare transcriptome-wide distributions of m⁶A between two different cell conditions. These methods map reads (red lines) immunoprecipitated by an m⁶A-binding antibody. m⁶A sites are often called based on whether there are a sufficient number of reads above a threshold. However, the number of reads that map to any m⁶A sites can be very variable, even between replicates (McIntyre et al., 2020). Thus, the same m⁶A site in one sample might be called in one sample, but not the other, if the number of reads just passes or misses the threshold. For this reason, the differences in the transcriptome-wide distribution of m⁶A in many experiments may have been highly overestimated.