[13]. Together these studies suggest that D1 receptor expression is regulated at the post-transcriptional level during brain develop- ment and under pathological conditions. The molecular mecha- nisms that mediate the post-transcriptional regulation of D1 receptor expression are not known. In this paper, we tested the hypotheses that the expression of D1 receptor is post-transcrip- tionally regulated during early postnatal mouse brain development and that the D1 receptor post-transcriptional regulation is mediated via trans-acting factors binding cis-elements in the 39- untranslated region (39UTR) of the D1 receptor mRNA. Using the mouse CATH.a-derived (CAD) catecholaminergic neuronal cell line, which endogenously expresses functional D1 dopamine receptor, we have previously shown multi-modal regulation of D1 receptor expression during serum withdrawal- induced neuronal differentiation [8]. In addition, the BDNF- mediated increase in endogenous D1 receptor expression in CAD cells also exhibits post-transcriptional regulation with the increase in D1 receptor protein being temporally delayed by greater than nine hours following the increase in D1 receptor mRNA levels [9]. Thus, the CAD cells are an ideal in vitro model to study the molecular mechanisms underlying post-transcriptional regulation of endogenously expressed D1 receptors. In this paper we demonstrate that the D1 receptor exhibits post-transcriptional regulation during postnatal mouse brain development and use the CAD cell line to identify the molecular mechanisms underlying D1 receptor post-transcriptional regulation. Using a systematic approach, we demonstrate that the D1 receptor 39UTR is necessary and sufficient for D1 receptor post-transcriptional regulation. We demonstrate for the first time that the microRNA, miR-142-3p, directly regulates D1 receptor post-transcriptional regulation in CAD cells and that its expression is inversely correlated to D1 receptor protein expression during postnatal mouse brain development. Furthermore, specific inhibition of endogenous miR-142-3p in CAD cells increases D1 receptor protein levels and enhances D1 receptor mediated-signaling. This study is the first to report that a noncoding RNA-mediated translational suppression mechanism regulates the expression of D1dopamine receptors. Materials and Methods Animals and Brain Tissue Harvest Male mice with a Swiss Webster/FVB genetic background were used in the study. The mice used in this study were obtained from locally bred animals kept on a 12:12 hour, light-dark schedule (lights on at 0800) and provided ad libitum food and water. The animal protocols were approved by the IACUC committee at UMDNJ-New Jersey Medical School. Whole brain was isolated, sectioned on a Vibratome and the dorsal striatum, including the caudate-putamen region punched out from the appropriate slices. The punches for RNA isolation were stored in RNAlaterH (Ambion) and those for protein analysis rapidly frozen in a dry ice-ethanol mixture and stored at 280uC. Cell Culture and Transfection CAD cells were maintained in DMEM/F12 media, 8% fetal calf serum (FCS) and 100 U/mL penicillin/streptomycin. CAD cells used in the experiments were plated and grown in either 6- or 12-well tissue culture plates. CAD cells were plated and grown for 24 hours or more in serum-containing media to about 60% confluence before transfection. Differentiation and transfection of CAD cells were done as described previously [8–10]. For transfection, the Lipofectamine 2000 (Invitrogen) transfection reagent and test plasmid DNA’s were mixed in OPTI-MEM D1 Receptor Expression Is Regulated by MicroRNA media and the mixture overlaid on non-differentiated CAD cells in serum media for six hours. After six hours, the media was replaced with fresh serum-containing or serum-free media and the cells harvested 48 hours later. The transfection efficiency was monitored by co-transfecting either a plasmid expressing the enhanced green fluorescent protein (EGFP) reporter gene or the FlagTM-tagged bacterial alkaline phosphatase gene (BAP-FlagTM). To inhibit microRNA function, anti-mirs that specifically targeted miR-142-3p (Ambion) were transfected at a concentration of 30 nM per well (for a 12-well plate). A FAM-labeled fluorescent anti-mir (Ambion) was used as a negative control. To knock-down Dicer levels in non-differentiated CAD cells, we transfected two different siRNAs that specifically targeted the mouse Dicer mRNA (Ambion) at a concentration of 10 nM per well (for a 12-well plate). A FAM-labeled fluorescent siRNA (Ambion) was used as a negative control. In functional experiments, endogenous miR-142- 3p was also inhibited using a miRZipTM anti-sense microRNA expressing plasmid with anti-miR-142-3p activity (System Biosci- ences, Mountain View, CA, USA). For these functional experi- ments we also generated and used a negative control empty miRZipTM vector plasmid in which the nucleotides encoding the anti-sense miR-142-3p were deleted. Cloning, Deletion and Mutagenesis The cloning of the 6400 bp mouse D1 receptor promoter has been previously described [9]. The mouse D1 receptor 39UTR region (the 1277 bp and 1684 bp fragments) was amplified using specific primers and a BAC construct containing the entire mouse D1 receptor gene (BAC clone address RP23-47M2; Invitrogen). The primers included Not I and HindIII/AflII/PmlI restriction sites which facilitated the cloning of the amplified D1 39UTR into the various reporter plasmids. The various deletion constructs were generated using PCR primer pairs that flanked the polyadenylation site within the 1277 bp D1 receptor 39UTR. The primers used for generating the deletion constructs also contained the above restriction enzyme sites to facilitate cloning of the products into the reporter plasmids. The D1 receptor 39UTR constructs with mutations in the microRNA binding sites were generated using a mutagenic primer with a KpnI restriction site that disrupted the individual microRNA seed recognition sequence (Figure S1). The three different microRNA binding site mutants were made in the context of a reporter construct that included the 1277 bp D1 receptor 39UTR. In addition, wild-type and mutant D1 receptor 39UTRs were individually subcloned into a reporter plasmid that included a heterologous bovine growth hormone 39UTR. All recombinant plasmids were sequenced and the wild type D1 receptor 39UTR sequence was found to match the sequence in the NCBI database. All plasmids were purified on two CsCl gradients prior to sequencing and transfection. Real-time Reverse Transcriptase PCR RNA isolation and RT-PCR was performed as described previously [8–10]. Briefly RNA was isolated using TrizolH reagent (Invitrogen) or the RNeasyH Mini Kit (Qiagen) according to the manufacturers’ instructions. RT reaction was carried out using Superscript III RT. PCR was performed using the Roche Light Cycler (Indianapolis, IN, USA) in either the SYBRH Green format, using primers and conditions described previously, or using gene-specific TaqManH gene expression assays (Applied Biosystems) [8–10]. The RT-PCR conditions were optimized as shown in Figure S2. Appropriate negative and positive controls were included in the RT-PCR experiments. For the SYBERH green method, the products were run on an agarose gel, stained PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49288