Distinct m6A and m1A Responses to Neuronal Depolarization in Cortical Neurons

Neuronal plasticity in response to external stimuli underlies learning and memory, and RNA methylation has emerged as a critical regulator of this process. Yet how different modifications contribute to activity-dependent regulation remains unclear. N1-Methyladenosine (m1A) and N6-methyladenosine (m6A) are methylation both occurred at adenosine, but at different positions. To compare m1A and m6A dynamics in primary cortical neurons during neuronal activation mimicked by KCl depolarization, we performed single-nucleotide resolution GLORI-seq and m1A MAP-seq while overall m6A levels were markedly reduced, m1A levels remained stable. m6A sites exhibited a shift from the canonical DRACH motif to AA-rich contexts, whereas m1A motifs were unchanged. Structure modeling revealed preferential localization of m1A to low-minimum free energy (MFE), loop-enriched regions, in contrast to a broader distribution of m6A across flexible structures. Integration with RNA-seq data further showed that transcripts harboring upregulated m1A sites displayed elevated expressions, particularly when these sites were embedded in loop-like RNA structures, suggesting a structural basis for m1A-mediated transcript enhancement. In contrast, m6A site changes were not associated with transcriptional differences, but their neighboring sequences were enriched for translation-related RNA-binding proteins. Together, these findings define a division of labor between adenosine methylations: m1A enhances a RNA output via structural encoding, while m6A modulates translation through dynamic RNA-binding protein interactions, offering a mechanistic framework for epitranscriptomic coordination during activity-dependent neuronal plasticity.