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.