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<p>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 <i>N</i><sup>1</sup>-methyladenosine (m<sup>1</sup>A). Initial transcriptome-wide maps reported thousands of internal m<sup>1</sup>A sites while later studies disputed these findings and showed that these sites lacked the expected m<sup>1</sup>A-induced misincorporations. More recently, an evolved reverse transcriptase that detects m<sup>1</sup>A&#xa0;was developed to resolve this issue and was used to map sites that again suggested that internal m<sup>1</sup>A 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&#xa0;detect modified nucleotides in RNA. Using SCARPET, we show that newly mapped internal m<sup>1</sup>A sites are not m<sup>1</sup>A, but&#xa0;instead contain inosine from A-to-I editing. However, <i>N</i>⁷-methylguanosine (m<sup>7</sup>G) sites, which are also controversial, were confirmed to occur at high stoichiometry. These results confirm that view that m<sup>1</sup>A is extremely rare and establish SCARPET as a robust strategy for validating putative&#xa0;modification sites.</p>

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

  • 期刊论文

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  • 发表日期：

    2026年6月23日

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  • Rohit Nalavade,
  • Aashiq H Mirza,
  • Samie R Jaffrey

摘要

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.