Differential Allelic Expression of Dopamine D1 Receptor Gene ( DRD1) Is Modulated by microRNA miR-504

Abstract

Previously, we reported that dopamine D1 receptor gene (DRD1) is associated with nicotine dependence (ND) and demonstrated that two alleles (A and G) of polymorphism rs686 in the 3'-untranslated region (3'UTR) of DRD1 are expressed differentially. However, the mechanism underlying the differential expression remains to be determined. We hypothesize that it is caused by miRNA targeting. We first used the MicroInspector algorithm to identify microRNAs (miRNAs) potentially targeting the rs686 polymorphism in the DRD1 3'UTR and then employed a luciferase reporter assay combined with site-directed mutagenesis to test the predicted miRNA targeting. We also examined the miRNA targeting of DRD1 with a gene expression assay. Of two miRNAs predicted by computational analyses, we found that miR-504, not miR-296, upregulated reporter luciferase activity and increased DRD1 expression by targeting the DRD1 3' UTR, whereas inhibition of miR-504, not miR-296, had the opposite effect. Furthermore, we showed that the direct binding of miR-504 to the DRD1 3'UTR, verified by site-directed mutagenesis, causes a significant expression difference between the two alleles. miR-504 up-regulates DRD1 expression by direct binding to the 3'UTR, which leads to differential allele-specific expression of DRD1.