MicroRNA 142-3p Mediates Post-Transcriptional Regulation of D1 Dopamine Receptor Expression Krishna E. Tobo´ n, Denis Chang, Eldo V. Kuzhikandathil* Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey, United States of America Abstract The D1 dopamine receptor subtype is expressed in the brain, kidney and lymphocytes. D1 receptor function has been extensively studied and the receptor has been shown to modulate a wide range of physiological functions and behaviors. The expression of D1 receptor is known to change during development, disease states and chronic treatment; however, the molecular mechanisms that mediate the changes in D1 receptor expression under these circumstances are not well understood. While previous studies have identified extracellular factors and signaling mechanisms regulating the transcription of D1 receptor gene, very little is known about other regulatory mechanisms that modulate the expression of the D1 receptor gene. Here we report that the D1 receptor is post-transcriptionally regulated during postnatal mouse brain development and in the mouse CAD catecholaminergic neuronal cell line. We demonstrate that this post-transcriptional regulation is mediated by a molecular mechanism involving noncoding RNA. We show that the 1277 bp 39untranslated region of D1 receptor mRNA is necessary and sufficient for mediating the post-transcriptional regulation. Using deletion and site-directed mutagenesis approaches, we show that the D1 receptor post-transcriptional regulation is specifically mediated by microRNA miR-142-3p interacting with a single consensus binding site in the 1277 bp 39untranslated region of D1 receptor mRNA. Inhibiting endogenous miR-142-3p in CAD cells increased endogenous D1 receptor protein expression levels. The increase in D1 receptor protein levels was biologically significant as it resulted in enhanced D1 receptor- mediated signaling, determined by measuring the activation of both, adenylate cyclase and, the dopamine- and cAMP- regulated phosphoprotein, DARPP-32. We also show that there is an inverse correlation between miR-142-3p levels and D1 receptor protein expression in the mouse brain during postnatal development. This is the first study to demonstrate that the post-transcriptional regulation of D1 receptor expression is mediated by microRNA-induced translational suppression. Citation: Tob o´n KE, Chang D, Kuzhikandathil EV (2012) MicroRNA 142-3p Mediates Post-Transcriptional Regulation of D1 Dopamine Receptor Expression. PLoS ONE 7(11): e49288. doi:10.1371/journal.pone.0049288 Editor: Erik C. Johnson, Wake Forest University, United States of America Received August 1, 2012; Accepted October 8, 2012; Published November 12, 2012 Copyright: 2012 Tobo´ n et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health grants R03DA026030 and R03DA026030-02S1 and a grant from the F. M. Kirby Foundation to EVK. KET is supported by a PhRMA Foundation pre-doctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kuzhikev@umdnj.edu Introduction The neurotransmitter dopamine binds and activates two major subfamilies of dopamine receptors. The D1-like subfamily includes D1 and D5 receptors and the D2-like subfamily includes D2, D3 and D4 receptors. The D1 dopamine receptor subtype is expressed at high levels in the basal ganglia and prefrontal cortex regions in humans and rodents [1]. In addition, several studies have shown that the D1 dopamine receptor is expressed in the kidneys [2] and lymphocytes [3]. D1 receptor signaling function has been extensively studied and it has been shown to activate adenylate cyclase and modulate ion channel function [4]. D1 receptors in the brain are involved in motor control and cognition and are essential for mediating addictive behaviors [1]. In the kidney, D1 receptors modulate the sodium-potassium ATPase and the sodium-hydro- gen exchanger and regulate diuresis and natriuresis [5]. Recently, peripheral dopamine acting via D1 receptor expressed on antigen- presenting dendritic cells and T-cells was shown to modulate the differentiation of various types of T-cells following immune activation [3]. Thus the D1 receptor is expressed in the periphery and the brain and plays an important role in many physiological and pathophysiological conditions. The expression of D1 receptor is plastic and its levels change during development, aging, pathological conditions and following chronic drug treatment [6,7]. However, the molecular mecha- nisms and extracellular factors that regulate the expression of the D1 receptor gene are not well understood. We have previously shown that the expression of D1 receptor can be regulated at the transcriptional, post-transcriptional and post-translational level during neuronal differentiation [8]. We have also shown that extracellular factors such as brain derived neurotrophic factor (BDNF), NT-3 and adenosine regulates D1 receptor expression at the transcriptional level [8–10]. In the developing rat brain, the expression of D1 receptor mRNA begins to increase around embryonic day 14 and reaches steady state expression level around postnatal day 5; in contrast, D1 receptor protein levels increase postnatally reaching peak values between postnatal day 7 and 14 [6,11]. This lack of correlation in expression of D1 receptor mRNA and protein is also seen during human brain development [12]. Interestingly, the expression of D1 receptor protein in lymphocytes also shows a similar age-dependent upregulation PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49288