M(6)A RNA Methylation-Based Epitranscriptomic Modifications in Plasticity-Related Genes via miR-124-C/EBPα-FTO-Transcriptional Axis in the Hippocampus of Learned Helplessness Rats

BACKGROUND: Impaired synaptic plasticity has been linked to dynamic gene regulatory network changes. Recently, gene regulation has been introduced with the emerging concept of unique N6-methyladenosine (m(6)A)-based reversible transcript methylation. In this study, we tested whether m(6)A RNA methylation may potentially serve as a link between the stressful insults and altered expression of plasticity-related genes. METHODS: Expression of plasticity genes Nr3c1, Creb1, Ntrk2; m6A-modifying enzymes Fto, methyltransferase like (Mettl)-3 and 14; DNA methylation enzymes Dnmt1, Dnmt3a; transcription factor C/ebp-α; and miRNA-124-3p were determined by quantitative polymerase chain reaction (qPCR) in the hippocampus of rats that showed susceptibility to develop stress-induced depression (learned helplessness). M(6)A methylation of plasticity-related genes was determined following m(6)A mRNA immunoprecipitation. Chromatin immunoprecipitation was used to examine the endogenous binding of C/EBP-α to the Fto promoter. MiR-124–mediated post-transcriptional inhibition of Fto via C/EBPα was determined using an in vitro model. RESULTS: Hippocampus of learned helplessness rats showed downregulation of Nr3c1, Creb1, and Ntrk2 along with enrichment in their m(6)A methylation. A downregulation in demethylating enzyme Fto and upregulation in methylating enzyme Mettl3 were also noted. The Fto promoter was hypomethylated due to the lower expression of Dnmt1 and Dnmt3a. At the same time, there was a lower occupancy of transcription factor C/EBPα on the Fto promoter. Conversely, C/ebp-α transcript was downregulated via induced miR-124-3p expression. CONCLUSIONS: Our study mechanistically linked defective C/EBP-α-FTO-axis, epigenetically influenced by induced expression of miR-124-3p, in modifying m(6)A enrichment in plasticity-related genes. This could potentially be linked with abnormal neuronal plasticity in depression.