D1 Receptor Expression Is Regulated by MicroRNA Figure 10. Expression level of miR-142-3p in postnatal striatal development is inversely correlated to D1 receptor protein expression. (A) Representative western blot which shows the level of D1 receptor protein in mouse brain striatum at P7, P14 and P30. Equal levels of total protein were loaded in each lane of the gel as evidenced by the equivalent amido black staining in the bottom panel. (B) Relative level of miR- 142-3p normalized to U6 small nuclear RNA in striatal tissue punches at P7, P14 and P30. The bars represent the mean values 6 s.e.m. (n = 3 to 4). *P,0.001, the level of miR-142-3p was significantly higher at P7 when compared to P14 and P30, One Way ANOVA, post hoc SNK test. doi:10.1371/journal.pone.0049288.g010 Discussion The neurotransmitter dopamine controls many different phys- iological and behavioral functions in mammals through receptors that are members of seven transmembrane-spanning G-protein coupled receptor family. The D1 dopamine receptor is one of the major subtypes of dopamine receptors and is widely expressed in the brain and periphery. While numerous studies have extensively characterized D1 receptor function, mechanisms and factors that regulate its expression are less well characterized. The results presented in this paper establishes post-transcriptional regulation as a significant mechanism that controls the expression of D1 dopamine receptor protein during postnatal mouse brain devel- opment (Fig. 1) and in the mouse CAD catecholaminergic cell line (Fig. 2). We demonstrated that the D1 receptor 39UTR was necessary and sufficient of mediating the post-transcriptional regulation (Figs. 3 and 4). The D1 receptor 39UTR has not been defined; however comparison of the mouse D1 receptor 39UTR, used in this study, to the rat and human D1 receptor 39UTR revealed two poly adenylation signal sites 714 and 1635 nucleotides downstream from the stop codon. Our results suggest that the 1277 nucleotides long D1 receptor 39UTR containing the first poly adenylation signal is necessary and sufficient for post- transcriptional regulation. Deletion and site-directed mutagenesis studies showed that the D1 receptor post-transcriptional regulation in CAD cells is mediated by a specific interaction of miRNA miR- 142-3p with a single cis-element adjacent to the first poly adenylation signal site in the D1 receptor 39UTR (Figs. 5, 6, and 7). Posttranscriptional regulation of many genes is mediated by miRNA. These ,22 nucleotide long endogenous RNA molecules are partially complementary to nucleotide sequences in the 39UTR of genes. By binding to the 39UTR with partial complementarity, miRNA molecules have been reported to repress translation of the mRNA or affect mRNA stability. Previous studies have shown that miRNAs modulate expression of various G-protein coupled receptors including the b-adrenergic receptor [15], adenosine A2A receptor [16], serotonin receptors [17] and estrogen receptor a [18]. Our results add D1 dopamine receptors to the growing list of G-protein coupled receptors whose expression is regulated by miRNAs. D1 receptors couple to activate adenylate cyclase, increasing intracellular cAMP levels. The increased cAMP activates the cAMP-dependent kinase which in turn phosphorylates the protein phosphatase DARPP-32. Activation of D1 receptors is known to increase phosphorylated DARPP-32 levels both in vitro and in vivo. The regulation of endogenous D1 receptor expression by miR- 142-3p is biologically significant as inhibition of miR-142-3p increased endogenous D1 receptor protein levels (Fig. 8) and enhanced D1 receptor signaling as evidenced by increased cAMP production and phospo-DARPP-32 levels (Fig. 9). Finally, we showed that the expression of miR-142-3p was inversely correlated to D1 receptor protein expression during postnatal mouse brain development (Fig. 10). To our knowledge this is the first report of a microRNA mediated translational suppression of any of the five dopamine receptor subtypes. One previous study showed that miR-504 specifically targets a polymorphic site in human D1 39UTR that is associated with nicotine dependence; however in that study miR-504 was found to increase, rather than decrease, the expression level of the human D1 receptor mRNA [19]. The identification of miR-142-3p as a regulator of D1 receptor expression was unexpected as the function of this microRNA has been previously described only in hematopoietic cells and lymphocytes were it was shown to modulate the differentiation of various classes of T-cells [20]. The expression of miR-142-3p is particularly high in the antigen-presenting dendritic cells [20]. Interestingly, lipopolysaccharide (LPS) treatment decreases the expression of miR-142-3p in dendritic cells, specifically relieving the translational repression of the interleukin-6 (IL-6) mRNA and increasing the level of secreted IL-6 [20]. Circulating levels of IL-6 is high in schizophrenic patients [21] and administration of IL-6 has been reported to increase stereotypic behavior in rodent models [22–24]. IL-6 along with TGF-b has also been shown to promote the differentiation of naive T-cells into the T-helper, Th- PLOS ONE | www.plosone.org 13 November 2012 | Volume 7 | Issue 11 | e49288