Elucidating the Requirements for microRNA-Mediated Upregulation Enables Reengineering of Upregulatory Sites

The post-transcriptional regulation of protein expression by miRNA has given rise to new technologies for therapeutic interventions and synthetic biology. To date, these technologies assume that miRNAs are repressors of protein expression; however, emerging evidence has shown that miRNA regulation of proteins is bidirectional. Although it is now clear that upregulation by miRNAs is a common event, the molecular requirements remain poorly characterized. To address this gap, we utilized the miRFluR assay, a genetically encoded dual-fluorescence reporter assay previously developed in our laboratory, to systematically explore upregulation. Using upregulation of the enzyme B3GLCT by miR-891b as a model system, we find that upregulation requires both specific sequence elements and RNA secondary structure. Mutagenesis and truncation analyses revealed that the effect is context-dependent and influenced by the local UTR structure. We also find that sites can be reengineered to be triggered by alternate miRNA, opening the door to synthetic biology manipulations. Our study provides a new perspective on upregulation by miRNA and sets the stage for further exploration of the bidirectional nature of miRNA action.