Epitranscriptomic shifts in M6A RNA methylation influencing transcriptional dynamics in the prefrontal cortex of chronic restraint stress rats

Chronic restraint stress (CRS) is a widely used model for investigating stress-induced molecular and neuronal changes. In this study, we examined transcriptome-wide m6A methylation in the prefrontal cortex of CRS rats to understand the molecular impact of stress. Elevated plasma corticosterone levels confirmed the physiological stress response in CRS rats. MeRIP-seq analysis identified 21,669 differentially methylated transcripts, with a predominant hypermethylation pattern (4,301 transcripts) compared to a smaller subset of hypomethylated transcripts (79). Chromosomal distribution revealed widespread hypermethylation across multiple chromosomes, with notable peaks on chromosomes 1, 3, and 10. Gene expression profiling indicated differential regulation of 1,424 genes, with 847 upregulated and 577 downregulated in CRS rats. Integration of m6A methylation and gene expression data revealed an inverse correlation, where hypermethylated transcripts were downregulated, suggesting a role for m6A in transcript stability and turnover. Functional analysis of hypermethylated transcripts highlighted enrichment in key neuronal processes, including synaptic plasticity, neurotransmitter signaling, and chromatin remodeling. Additionally, the 3′UTR of coding transcripts exhibited enriched m6A methylation marks, suggesting a regulatory role in mRNA stability and translation efficiency. RNA level expression analysis revealed significant downregulation of key m6A methylation-related enzymes (METTL3, METTL14, and ALKBH5), further supporting m6A dysregulation under CRS. Pathway analysis underscored the involvement of differentially methylated transcripts in RNA metabolism, chromatin remodeling, and neurobiological pathways linked to stress-related psychiatric disorders. Altogether, the study provides insight into the epitranscriptomic mechanisms underlying stress responses and their implications in neuropsychiatric disorders such as major depression.