Analyzing the miRNA regulatory landscape of OGT identifies evolutionarily conserved upregulation.

O-GlcNAc transferase (OGT) is the key enzyme involved in post-translationally modifying cytoplasmic and nuclear proteins with O-GlcNAc. Maintenance of cellular O-GlcNAcylation levels is critical to cell health and requires precise transcriptional and post-transcriptional control. Herein we examine the miRNA regulation of OGT by the human miRNAome using our high-throughput miRFluR assay. We found >200 miRNA regulators of OGT, including 17 down- and 15 upregulatory miRNAs previously identified in CLIP datasets. We validated the impact of select miRNA on OGT and O-GlcNAc levels using both miRNA mimics and inhibitors that reduce endogenous miRNA levels. We focused our studies on two miRNA families, the downregulatory let-7 family and the upregulatory miR-148/152 family. For the let-7 family, we found that only let-7a-3p and let-7g-3p strongly downregulated OGT. Downregulation required two seed-dependent binding sites. Evolutionary analysis found that the more recent of the two sites emerged in placental mammals. A similar conservation pattern was observed for the site of regulation by the miR-148/152 family, which was previously identified in CLIP datasets. All three miRNA in this family upregulated OGT. Phylogenetic analysis revealed that this upregulatory site has been conserved for the past 98.7 million years. The emergence of these regulatory sites correlates with that of disease states that both OGT and the miRNA are known to impact. Overall, our results provide important insights into OGT, miRNA regulation and conservation through evolution.