Small Molecule-Inducible and Photoactivatable Cellular RNA N1-Methyladenosine Editing

N1-methyladenosine (m¹A) modification is one of the most prevalent epigenetic modifications on RNA. Given the vital role of m¹A modification in RNA processing such as splicing, stability and translation, developing a precise and controllable m¹A editing tool is pivotal for in-depth investigating the biological functions of m¹A. In this study, we developed an abscisic acid (ABA)-inducible and reversible m¹A demethylation tool (termed AI-dm¹A), which targets specific transcripts by combining the chemical proximity-induction techniques with the CRISPR/dCas13b system and ALKBH3. We successfully employed AI-dm¹A to selectively demethylate the m¹A modifications at A8422 of MALAT1 RNA, and this demethylation process could be reversed by removing ABA. Furthermore, we validated its demethylation function on various types of cellular RNAs including mRNA, rRNA and lncRNA. Additionally, we used AI-dm¹A to specifically demethylate m¹A on ATP5D mRNA, which promoted ATP5D expression and enhanced the glycolysis activity of tumor cells. Conversely, by replacing the demethylase ALKBH3 with methyltransferase TRMT61A, we also developed a controllable m¹A methylation tool, namely AI-m¹A. Finally, we caged ABA by 4,5-dimethoxy-2-nitrobenzyl (DMNB) to achieve light-inducible m¹A methylation or demethylation on specific transcripts. Collectively, our m¹A editing tool enables us to flexibly study how m¹A modifications on specific transcript influence biological functions and phenotypes.