¹University of Alabama at Birmingham, Psychiatry, Birmingham, United States

Background: Increasing evidence suggests that fine-tuning of transcriptional regulation by gene x environment interaction is central to the etiology of major depressive disorder (MDD). In this regard, the concept of post-transcriptional gene regulation through “epitranscriptomic” mechanism is gaining traction, indicating their involvement not only in the regulation of transcript abundance but also their ability to impact maturity, stability, localization, and, most importantly, availability of “select” gene transcripts to protein translation despite varying transcription rates. This regulatory mechanism has the distinct ability to quickly respond to external stimuli, fine-tune protein accessibility, and execute localized control, which is critical to stimulus-dependent gene expression.

Aims & Objectives: In this study, we examined if m6A methylation-based mRNA modification and consequent expression of genes participate in MDD pathogenesis.

Method: Dorsolateral prefrontal cortex (dlPFC) from 49 non-psychiatric controls and 49 MDD subjects were used to measure m6A methylation. Nuclei from dlPFC were enriched to perform RNA extraction. Two hundred nt long random RNA fragments were produced, and fragmented RNAs were denatured. For methylated RNA immunoprecipitation, the anti-m6A antibody was used for cross-linking. cRNAs and immunoprecipitated RNAs were amplified and labeled separately with Cy5 and Cy3 fluorescent dyes. Raw intensities of Cy5-labeled RNA and Cy3-labeled RNA were normalized with an average log2-scaled spike-in RNA control. Differentially, m6A-methylated RNAs were identified by filtering with the fold change and two-tailed Student’s t-test. Fold-change ≥1.5 or ≤0.7 and P<0.05 were used to determine the differentially m6A methylated RNAs. Gene Ontology (GO) term enrichment analyses were performed for biological processes, cellular components, and pathways.

Results: Based on 53135 probe sets, 309 transcripts were identified as significantly hypermethylated in the MDD compared to the control group. On the other hand, 9174 transcripts were significantly hypomethylated. Chromosomes 9, 13, 16, and 22 were found to code low to moderate amounts of hypermethylated transcripts. Most of the methylation sites were associated with the coding region (CDS) of mRNA transcripts in contrast to the two untranslated regions (5’ and 3’UTR) flanking the CDS. Sequence motif analyses revealed the enrichment of the m6A consensus motif RRACH in all samples. Based on sequence conservation patterns, two high consensus motifs, AAGA and ACCCA were identified. Prediction of GO, based on the identified genes using the Panther classification system, identified significant functional enrichment (following FDR correction and Fisher exact test) for neuron development, trans-synaptic signaling, synaptic transmission, axonogenesis, neuron projection development, and nervous system development. KEGG analysis further revealed pathways, including signal transduction, immune system processing, and protein degradation. These pathways represented diverse molecular roles related to MDD, which included olfactory transduction, neuroactive ligand-receptor interaction, and signaling pathways such as PI3K-Akt, MAPK, Wnt, TNF, and nuclear factor-kappa B (NF-κB).

Discussion & Conclusions: The present study, for the first time, reports the phenomenon of RNA methylation modification in the context of MDD. It also unfolds the potential impact of this modification in understanding the impeded function of transcripts involved in neural plasticity-related pathways.