Validation of the mRNA epitranscriptome: SCARPET reveals that mapped m¹A sites are inosine

A major goal in epitranscriptomics is to define the diversity of modified nucleotides in mRNA. Most candidate sites are discovered using transcriptome-wide mapping methods that rely on antibody enrichment or modification-induced misincorporations during cDNA synthesis. However, these maps are rarely verified biochemically, and in some cases have yielded conflicting conclusions, most notably for N¹-methyladenosine (m¹A). Initial transcriptome-wide maps reported thousands of internal m¹A sites while later studies disputed these findings and showed that these sites lacked the expected m¹A-induced misincorporations. More recently, an evolved reverse transcriptase that detects m¹A was developed to resolve this issue and was used to map sites that again suggested that internal m¹A might be widespread. These divergent results underscore the need for biochemical testing of proposed sites. Here, we describe protocols for SCARPET (site-specific cleavage and radiolabeling followed by purification, exonuclease digestion, and thin-layer chromatography) to rapidly and efficiently detect modified nucleotides in RNA. Using SCARPET, we show that newly mapped internal m¹A sites are not m¹A, but instead contain inosine from A-to-I editing. However, N⁷-methylguanosine (m⁷G) sites, which are also controversial, were confirmed to occur at high stoichiometry. These results confirm that view that m¹A is extremely rare and establish SCARPET as a robust strategy for validating putative modification sites.