EGFP reporter protein in transfected CAD cells. The level of EGFP reporter mRNA generated by the 6.4 D1 and 6.4 D1+39UTR constructs under either nondifferentiated or differen- tiated conditions were equal (Fig. 3B and 3C), suggesting that the introduction of the D1 receptor 39UTR into the 6.4 D1 EGFP reporter construct had no effect on the level of steady-state EGFP reporter mRNA. In contrast to the results with mRNA, the levels of EGFP reporter protein from the 6.4 D1 and 6.4 D1+39UTR constructs were significantly different. Figures 3D and 3E show that introduction of the D1 receptor 39UTR significantly repressed the expression of EGFP reporter protein in both non-differentiated and differentiated CAD cells by greater than 15-fold. Together the results in Figure 3 suggest that sequences in the D1 receptor 39UTR represses translation. Analysis of the nucleotide sequences downstream of the D1 receptor stop codon identified a second poly adenylation signal downstream of the first poly adenylation signal (Figure S3A). We compared the D1 receptor post- transcriptional regulation in reporter constructs containing D1 receptor 39UTR that was either 1277 bp or 1684 bp long and found that both 39UTR’s suppressed the translation of the EGFP reporter protein equally (Figure S3B). This suggested that the D1 receptor post-transcriptional regulation is mediated by sequences in the 1277 bp D1 receptor 39UTR proximal to the stop codon. We next determined if the D1 receptor 39UTR was necessary and sufficient to repress the translation of a reporter mRNA that was generated from a heterologous promoter such as the cytomegalovirus (CMV) promoter. We introduced the 1277 bp D1 receptor 39UTR region into the pCMV b-galactosidase reporter construct (Fig. 4A). The resultant pCMV b-gal+D1 39UTR and the parental pCMV b-gal constructs were indepen- dently transfected into non-differentiated CAD cells along with a plasmid expressing the EGFP as a control for transfection efficiency. Forty eight hours after transfection, cells were harvested and b-galactosidase reporter mRNA and enzyme activity deter- mined (Fig. 4B and 4C). The b-galactosidase reporter and EGFP mRNA levels were measured using real time RT-PCR. The b- galactosidase reporter enzyme activity was detected using CPRG as a substrate. The results in Figure 4 show that the b-galactosidase reporter mRNA levels in cells transfected with pCMV b-gal and pCMV b-gal+D1 39UTR were identical. Yet the b-galactosidase activity (Fig. 4C) levels were reduced , 20-fold in cells transfected with pCMV b-gal+D1 39UTR construct. These results strongly suggest that, in CAD cells, the D1 receptor 39UTR is necessary and sufficient to suppress the translation of mRNA generated from a heterologous promoter. Taken together, these data strongly suggest that the D1 receptor 39UTR is necessary and sufficient for the post-transcriptional regulation of D1 receptor expression. MicroRNA 142-3p Directly Mediates the D1 Receptor Post-transcriptional Regulation in CAD Cells The D1 receptor post-transcriptional regulation could be mediated by decreased mRNA stability, decreased protein stability or translational repression. The above results showing equivalent steady state mRNA levels of the endogenous D1 receptor and the two reporter genes (EGFP and b-galactosidase), taken together with our previous study showing no difference in D1 receptor mRNA stability in nondifferentiated and differentiated CAD cells [8], suggested that D1 receptor post-transcriptional regulation was not mediated by decreased mRNA stability. Similarly the observation that D1 receptor 39UTR post-transcriptionally regulates endogenous D1 receptor protein as well as two dissimilar reporter proteins (EGFP and b-galactosidase) suggested that decreased protein stability was unlikely to mediate D1 receptor post-transcriptional regulation. To identify the molecular mech- D1 Receptor Expression Is Regulated by MicroRNA anism mediating translational repression, we created deletion constructs using the pCMV b-gal+D1 39UTR parent construct in which the proximal and distal sequences flanking the poly adenylation site in the D1 receptor 39UTR were deleted. The constructs with the D1 receptor 39UTR deletions were transfected into non-differentiated CAD cells and b-galactosidase mRNA and activity measured 48 hours post-transfection. The results in Figure 5A show that the smallest deletion construct (pCMV b- gal+D1 39UTR [94 bp]) continued to express low levels of b- galactosidase activity, suggesting that the 94 bp D1 receptor 39UTR was necessary and sufficient for post-transcriptional regulation. There was no significant difference in the b-galacto- sidase mRNA levels expressed from these deletion constructs (Figure S4A and S5). Sequence analysis of the 94 bp D1 receptor 39UTR fragment revealed a consensus binding site for the highly conserved microRNA, miR-142-3p (Fig. 5B). To determine if the translational repression of D1 receptor was mediated by microRNA, we first used siRNAs to reduce the expression of Dicer, the protein involved in the processing of microRNA precursors. The results in Figure 6A and 6B show that the transient knockdown of Dicer expression in nondifferentiated CAD cells significantly relieved the translational repression of the b-galactosidase reporter activity expressed from the pCMV b- gal+D1 39UTR construct. Control experiments showed that the siRNA-mediated knockdown of Dicer expression had no effect on b-galactosidase expression from the parent pCMV b-gal construct (data not shown). These results suggested that D1 receptor post- transcriptional regulation might be mediated by Dicer-processed microRNAs. Given the results of the deletion experiments in Figure 5A and the Dicer knock-down experiments in Figure 6, we generated pCMV b-gal+D1 39UTR (1277 bp) constructs in which we specifically mutated nucleotides in the 1277 bp D1 receptor 39UTR that were complementary to the seed sequence of microRNA miR-142-3p (Fig. 5B) and two other microRNAs (miR-294/295 and 10a*). The latter microRNAs have conserved consensus binding sites in the D1 receptor 39UTR and served as negative controls. Rather than deleting the entire miRNA binding site, we took a conservative approach, and mutated the seed sequence binding sites for the three microRNAs (Figure S1) in the D1 receptor 39UTR. The individual mutated pCMV b-gal+D1 39UTR constructs were transfected into non-differentiated and differentiated CAD cells and b-galactosidase mRNA and activity measured 48 hours post-transfection. The results show that mutation of the miR-142-3p recognition site in the 1277 bp D1 receptor 39UTR, but not the 10a* or 294/295 sites, significantly increased the b-galactosidase reporter enzyme activity in both non-differentiated and differentiated CAD cells. Control experi- ments showed that mutation of these binding sites had no significant effect on the b-galactosidase mRNA levels from the mutated pCMV b-gal+D1 39UTR constructs (Figure S4B). To further confirm these results and to determine if the miR-142-3p translation repression was maintained in the context of a heterologous 39UTR, we cloned the 1277 bp wild type and the three mutated D1 receptor 39UT regions into a b-galactosidase expression plasmid with a heterologous bovine growth hormone (BGH) poly adenylation site (Fig. 7A). The resultant constructs were transfected into non-differentiated CAD cells and the b- galactosidase activity measured 48 hours post-transfection. The results confirmed that the D1 receptor 39UTR repressed the translation of the b-galactosidase reporter protein in the presence of a heterologous poly adenylation site and this repression was abolished by specifically mutating the recognition site for miR- 142-3p (Figure 7B). PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e49288