D1 Receptor Expression Is Regulated by MicroRNA Figure 4. The D1 39UTR is necessary and sufficient for D1 receptor post-transcriptional regulation. (A) Schematic representation of reporter plasmids with the b-galactosidase (b-gal) reporter gene regulated by the constitutively active cytomegalovirus (CMV) promoter. In one of the constructs, the recombinant 39UTR with the SV40 polyadenylation (poly A) sequence was replaced by the 1277 bp D1 receptor 39UTR. (B) Non differentiated CAD cells were transfected with either control pcDNA plasmid (Mock), the positive control pCMV b-gal plasmid with SV40 poly A (pCMV b-gal) or the plasmid containing CMV b-gal and the 1277 bp D1 receptor gene 39UTR (pCMV b-gal+D1 39UTR). The cells were also co-transfected with a plasmid expressing EGFP to control for transfection efficiency. The b-gal and EGFP mRNA levels (upper panels) were measured using real time RT- PCR and the products were run out on an agarose gel. Also shown is the EGFP protein level in the transfected cells. (C) The b-gal reporter enzyme activity was normalized to total protein amounts. The bars represent the mean values 6 s.e.m. (n = 3). The enzyme activity was significantly reduced in cells transfected with CMV b-gal+D1 39UTR compared to cells transfected with parental CMV b-gal plasmid (*, P,0.001, two-tailed pair-wise Student’s t-test). doi:10.1371/journal.pone.0049288.g004 To determine if miR-142-3p modulates the expression of endogenous D1 receptor protein in CAD cells, we transiently- transfected anti-mirs that specifically targeted miR-142-3p and measured endogenous D1 receptor protein levels. The results in Figure 8 show that anti-mir mediated inhibition of miR-142-3p significantly increased D1 receptor protein levels in non-differen- tiated and differentiated CAD cells when compared to a FAM- labeled negative control anti-mir. Taken together, these results strongly suggest miR-142-3p directly regulates the D1 receptor 39UTR-mediated translational repression of D1 receptor expres- sion in CAD cells. Antisense-mediated Inhibition of miR-142-3p in CAD Cells Enhances D1 Receptor Signaling Function The experiments in Figures 6, 7 and 8 suggested that mutating the miR-142-3p or knocking down miR-142-3p levels using anti- mirs increases the protein expression ,80%. To determine if this increase was biologically significant, we next determined the effect of inhibiting endogenous miR-142-3p on endogenous D1 receptor signaling function in CAD cells. To decrease the levels of miR- 142-3p, in these experiments we used the miRZipTM anti-sense 142-3p plasmid construct. Figure 9A shows that co-transfection of miRZipTM anti 142-3p plasmid with the pCMV b-gal+D1 39UTR Figure 5. A 94 bp fragment of the D1 receptor 39UTR is necessary and sufficient for post-transcriptional regulation. (A) Relative b- galactosidase activity, normalized to total protein, in non-differentiated CAD cells transfected with the parent pCMV b-gal plasmid, pCMV b-gal+D1 39UTR or the various pCMV b-gal+D1 39UTR constructs with deletions in the 39UTR. The schematic representation of the 1277 bp D1 39UTR is shown with the white regions representing the deleted segments. All deletion constructs retained the poly adenylation signal (p(A)S). The b-gal reporter enzyme activity was normalized to total protein amounts. The bars represent the mean values 6 s.e.m. (n = 5). *P,0.028, the enzyme activity was significantly reduced in cells transfected with CMV b-gal+D1 39UTR and the various deletions constructs when compared to cells transfected with parental CMV b-gal plasmid, Kruskal-Wallis One Way ANOVA on Ranks, post hoc SNK test. (B) The nucleotide sequence of the 94 bp D1 receptor 39UTR that retained post-transcriptional regulation. The poly adenylation signal and the consensus binding site for microRNA miR-142-3p are shown. The * indicate the nucleotides in the seed recognition sequence that was mutated using site directed mutagenesis for the experiment in Figure 6C and 6D. doi:10.1371/journal.pone.0049288.g005 PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e49288