M6A RNA Methylation-Based Epitranscriptomic Modifications in Plasticity-Related Genes via miR-124-C/EBPα-FTO-Transcriptional Axis in the Hippocampus of Learned Helplessness Rats

Abstract

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 m 6A-based reversible transcript methylation. In this study, we tested whether m 6A 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, Mettl3, Mettl14; DNA methylation enzymes Dnmt1, Dnmt3a; transcription factor C/ebp-α; and miRNA-124-3p were determined by qPCR in the hippocampus of rats who showed susceptibility to develop stress-induced depression (learned helplessness, LH). M 6A methylation of plasticity-related genes was determined following m 6A mRNA immunoprecipitation. Chromatin immunoprecipitation was used to examine the endogenous binding of C/EBP-αto the Fto promoter. MiR-124-mediated post-transcriptional inhibition of Fto via C/EBPα was determined using an in-vitro model. Results Hippocampus of LH rats showed downregulation of Nr3c1, Creb1, and Ntrk2 along with enrichment in their m 6A 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 6A enrichment in plasticity-related genes. This could potentially be linked with abnormal neuronal plasticity in depression.

Effect of RNA m6A methyltransferase activation by a low molecular weight compound on anxiety- and depression-related behaviours, monoamine neurochemistry and striatal gene expression in the rat

“…The copyright holder for this preprint this version posted January 8, 2024. ; https://doi.org/10.1101/2024.01.08.574593 doi: bioRxiv preprint a positive correlation of m 6 A methylation and the methyltransferase Mettl3 levels with ability of contextual fear discrimination and demonstrated that expression of Mettl3 in hippocampal glutamatergic neurons is crucial for this type of learning and memory (Chang et al, 2022). In learned helpless rats the expression of several so-called plasticity genes in hippocampus was downregulated along with enrichment in their m 6 A methylation (Roy et al, 2022). In summary, RNA m 6 A methylation serves as a crucial regulation mechanism at the epitranscriptomic level to shape synaptic plasticity and behaviour (Malovic and Pandey, 2023) and involvement of RNA m 6 A methylation appears in multiple behavioural conditions relevant to anxiety, but so far evidence for a role of m 6 A methylation has remained correlational.…”