Abstract
BACKGROUND: Epitranscriptomic modifications modulate diverse biological processes like regulation of gene expression, abundance, location and function. N6-methyladenosine (m (6) A) methylation has been shown to regulate various diseases, including cancer and inflammation. While there is evidence that m (6) A modification is functionally relevant in neural development and differentiation, the role of m (6) A modification in HIV neuropathogenesis is unknown. METHODS: Here, we used anti-N6-methyladenosine (m (6) A) antibody immunoprecipitation and microarray profiling to identify m (6) A modifications in miRNAs in basal ganglia (BG) of Rhesus macaques (RMs) that were uninfected (VEH) and SIV-infected on combination anti-retroviral therapy (cART) and either VEH-treated (VEH/SIV/cART), or THC:CBD-treated (THC:CBD/SIV/cART). Ingenuity pathway analysis was conducted to understand the biological implications of miRNA m (6) A methylation in HIV neuropathogenesis. Finally, to understand the functional significance of m (6) A modifications in miRNAs, we overexpressed FAM-labeled wild-type or m (6) A-modified miR-194-5p in SCC-25 cells and determined its impact on the expression of its target, STAT1, an interferon-stimulated transcription factor known to drive persistent neuroinflammation in several neurodegenerative diseases. RESULTS: HIV/SIV infection promoted an overall hypomethylated miRNA m (6) A profile. While the overall hypomethylated m (6) A profile was not significantly impacted by THC:CBD, specific miRNAs predicted to target proinflammatory genes showed markedly reduced m (6) A methylation levels compared to the VEH-treated RMs. Additionally, specific BG tissue miRNAs bearing m (6) A epi-transcriptomic marks were transferred and detected in BG-derived extracellular vesicles. Mechanistically, the DRACH motif in the seed region of miR-194-5p was significantly m (6) A hypomethylated in THC:CBD/SIV/cART RMs. In SCC-25 cells, unlike wild-type miR-194-5p, transfected m (6) A-modified miR-194-5p mimics failed to downregulate STAT1 protein expression. Further, compared to VEH/SIV/cART RMs, THC:CBD administration significantly reduced m (6) A methylation of 44 miRNAs directly involved in regulating CNS network genes. CONCLUSIONS: These results underscore the need for investigating the qualitative, and posttranscriptional modifications in RNA along with the more traditional, quantitative alterations in pathological conditions or in response to disease modifying treatments. Our findings indicate that m (6) A epitranscriptomic marks in the seed nucleotide region can impair miRNA function and that cannabinoids may preserve it by reducing m (6) A methylation levels. Finally, these findings provide a novel mechanistic (miRNA m (6) A hypomethylation) explanation underlying the anti-neuroinflammatory effects of phytocannabinoids in HIV/SIV infection.