D1 Receptor Expression Is Regulated by MicroRNA Figure 1. D1 dopamine receptor exhibits post-transcriptional regulation during early postnatal mouse brain development. (A) Representative RT-PCR gel showing D1 receptor and internal control GAPDH mRNA levels in mouse brain striatum isolated from new born animals (P0) through postnatal day 14 (P14). The real-time RT-PCR was performed using the SYBRH green method and the reactions stopped in the exponential phase. (B) Cumulative data showing D1 receptor mRNA levels normalized to GAPDH internal control mRNA levels in the striatal region isolated from mice that were P0 through P14. The data are from real-time RT-PCR experiments using TaqManH probes specific for mouse D1 receptor and GAPDH. (C) Representative western blot showing D1 receptor protein levels in mouse brain striatum isolated from animals that were P0 through P14. The multiple bands correspond to different glycosylated forms of the D1 receptor [30,31]. The lower panel shows amido black staining of the membrane in the upper panel and shows the total protein loaded in each lane. (D) Cumulative data showing D1 receptor protein levels normalized to total protein. The plotted bars represent the mean values 6 s.e.m. (n = 5). *P,0.002, significant difference between P14 and all other ages, Kruskal- Wallis One Way ANOVA on Ranks, post hoc SNK test. doi:10.1371/journal.pone.0049288.g001 the D1 receptor mRNA [9]. Separately, we also cloned the 1277 bp 39UTR present in the D1 receptor mRNA. The 6.4 kb upstream D1 promoter region including the 59UTR (6.4 D1) was fused to an EGFP reporter gene (Fig. 3A). In a second construct, we subcloned the 1277 bp 39UTR of the D1 receptor downstream of the EGFP reporter gene, replacing the recombinant SV40 39UTR present in the vector of the 6.4 D1 construct (6.4 D1+39UTR; Fig. 3A). The 1277 bp 39UTR of the D1 receptor gene contains a single polyadenylation signal (AAUAAA). The two constructs were separately transfected into CAD cells along with a bacterial alkaline phosphatase (BAP)-FlagTM construct to serve as an internal control for transfection efficiency. Twelve hours after transfection, half of the transfected cells were switched to the serum-free differentiation media to induce CAD cell differentia- tion. All cells were harvested 48 hours later and the level of EGFP reporter mRNA, BAP-FlagTM mRNA, endogenous D1 receptor mRNA and 18S ribosomal RNA levels were measured using real- time RT-PCR with Syber greenH. The PCR reactions were stopped in the exponential phase and the products run out on a gel. In parallel, half of the harvested cells were used for protein analysis by western blot using antibodies specific for the EGFP protein and the FlagTM epitope. The results in Figure 3B and 3C show that differentiation of CAD cells increased expression levels of not only the endogenous D1 receptor mRNA, but also the EGFP reporter mRNA generated from the transfected 6.4 D1 and 6.4 D1+39UTR constructs. These results suggest that the 6.4 kb D1 promoter region in the EGFP reporter construct contains the sequences required for the differentiation-induced increase in D1 receptor mRNA. In addition, the presence of the D1 receptor 39UTR in the reporter construct did not affect its ability to increase mRNA expression following differentiation. To evaluate the ability of the reporter construct to mimic the post-transcriptional regulation of the endogenous D1 receptor gene, we compared the expression of EGFP reporter mRNA to PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49288