Neuronal Depolarization Induced RNA m⁵C Methylation Changes in Mouse Cortical Neurons

Neuronal activity is accomplished via substantial changes in gene expression, which may be accompanied by post-transcriptional modifications including RNA cytosine-5 methylation (m⁵C). Despite several reports on the transcriptome profiling of activated neurons, the dynamics of neuronal mRNA m⁵C modification in response to environmental stimuli has not been explored. Here, we provide transcriptome-wide maps of m⁵C modification, together with gene expression profiles, for mouse cortical neurons at 0 h, 2 h, and 6 h upon membrane depolarization. Thousands of differentially expressed genes (DEGs) were identified during the neuronal depolarization process. In stimulated neurons, the majority of early response genes were found to serve as expression regulators of late response genes, which are involved in signaling pathways and diverse synaptic functions. With RNA bisulfite sequencing data, a union set of 439 m⁵C sites was identified with high confidence, and approximately 30% of them were shared by neurons at all three time points. Interestingly, over 41% of the m⁵C sites showed increased methylation upon neuronal activation and were enriched in transcripts coding for proteins with synaptic functions. In addition, a modest negative correlation was observed between RNA expression and methylation. In summary, our study provided dynamic transcriptome-wide landscapes of RNA m⁵C methylation in neurons, and revealed that mRNA m⁵C methylation is associated with the regulation of gene expression.