High-Throughput Analysis Reveals miRNA Upregulating α-2,6-Sialic Acid through Direct miRNA–mRNA Interactions

[Image: see text] Chemical biology has revealed the importance of sialic acids as a major signal in physiology and disease. The terminal modification α-2,6-sialic acid is controlled by the enzymes ST6GAL1 and ST6GAL2. Dysregulation of this glycan impacts immunological recognition and cancer development. microRNAs (miRNA, miR), noncoding RNAs that downregulate protein expression, are important regulators of glycosylation. Using our recently developed high-throughput fluorescence assay (miRFluR), we comprehensively mapped the miRNA regulatory landscape of α-2,6-sialyltransferases ST6GAL1 and ST6GAL2. We found, contrary to expectations, the majority of miRNAs upregulate ST6GAL1 and α-2,6-sialylation in a variety of cancer cells. In contrast, miRNAs that regulate ST6GAL2 were predominantly downregulatory. Mutational analysis identified direct binding sites in the 3′-untranslated region (UTR) responsible for upregulation, confirming it is a direct effect. The miRNA binding proteins AGO2 and FXR1 were required for upregulation. Our results upend common assumptions surrounding miRNA, arguing that upregulation by these noncoding RNA is common. Indeed, for some proteins, upregulation may be the dominant function of miRNA. Our work also suggests that upregulatory miRNAs enhance overexpression of ST6GAL1 and α-2,6-sialylation, providing another potential pathway to explain the dysregulation observed in cancer and other disease states.