SCARPET: site-specific quantification of methylated and nonmethylated adenosines reveals m⁶A stoichiometry

Abstract

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. Using SCARPET, we measure exact m⁶A stoichiometries in specific mRNAs and show that Zika genomic RNA lacks m⁶A at previously mapped sites. 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.