SCARPET: Site-specific quantification of methylated and non-methylated adenosines reveals m6A stoichiometry.

Overview

m6A has different stoichiometry at different positions in different mRNAs. However, the exact stoichiometry of m6A is difficult to measure. Here we describe SCARPET (site-specific cleavage and radioactive-labeling followed by purification, exonuclease digestion, and thin-layer chromatography), a simple and streamlined biochemical assay for quantifying m6A at any specific site in any mRNA. SCARPET involves a site-specific cleavage of mRNA immediately 5' of an adenosine site in an mRNA. This site is radiolabeled with 32P, and after a series of steps to purify the RNA and to remove nonspecific signals, the nucleotide is resolved by TLC to visualize A and m6A at this site. Quantification of these spots reveals the m6A stoichiometry at the site of interest. SCARPET can be applied to poly(A)-enriched RNA, or preferably purified mRNA, which produces more accurate m6A stoichiometry measurements. We show that sample processing steps of SCARPET can be performed in a single day, and results in a specific and accurate measurement of m6A stoichiometry at specific sites in mRNA. SCARPET will be useful for testing specific sites for their m6A stoichiometry and to assess how m6A stoichiometry changes in different conditions and cellular contexts.