Selective detection of m6A derived from mRNA using the Phospho-tag m6A assay

N6-methyladenosine (m6A) is a modified nucleotide found in mRNA, ribosome RNA (rRNA) and small nuclear RNA (snRNA). m6A in mRNA has important roles in regulating mRNA stability, splicing, and other processes. Numerous studies have described m6A as a dynamic modification using mass spectrometry-based quantification of m6A in mRNA samples prepared from different cellular conditions. However, these results have been questioned based on the finding that the mRNA purification protocols often result in varying levels of rRNA contamination. Additionally, mRNA purification protocols disproportionately enrich for the 3 ends of mRNA, a region that is enriched in m6A. To address these problems, we developed the Phospho-tag m6A assay, a highly efficient method for quantifying m6A specifically from mRNA. In this assay, a series of selective RNase digestion steps is performed, which results in m6A from rRNA and snRNA being liberated as m6A monophosphate, while m6A from mRNA is mostly liberated as m6A nucleoside. m6A levels are normalized to transcript levels, using m7G monophosphate liberated by yDcpS decapping enzyme as a surrogate for mRNA levels. Notably, this approach uses total cellular RNA, rather than purified mRNA, which simplifies the steps for m6A detection and overcomes the 3-end biases associated with mRNA purification. Overall, the Phospho-tag m6A provides a simple and efficient method for quantification of mRNA-derived m6A from total RNA samples.