Impact of miR-495 vs. HuD in the Control of Addiction-Related Genes and Behavior

描述（由申请人提供）：控制可卡因的动机对于成功长期治疗可卡因成瘾至关重要，这可能需要逆转药物诱导的基因表达变化。尽管转录后机制在基因表达调控中起着至关重要的作用，但其在药物滥用中的作用却很少受到关注。RNA结合蛋白和microRNA作为控制基因表达的主开关，mRNA稳定性估计控制约20%的脑表达基因。我们的研究表明，RNA结合蛋白HuD和microRNA miR-495在控制成瘾相关基因的表达和行为中起着相反的作用：1）它们被预测与mRNA中相同的富含GU的序列结合; 2）它们的结合位点在成瘾相关基因（ARG）数据库的转录本中过度表达; 3）它们显示出可卡因的差异调节，其中miR-495下调，HuD上调; 4）这些分子的体外操作对其两个靶基因BDNF和arc的表达产生相反的影响;和5）最重要的是，这些分子的体内操作显示出对可卡因动机的对比效果。基于这些结果，我们假设HuD和miR-495竞争与相同序列的结合，以控制ARG的表达和相反方向的药物动机。为了验证这一假设，在CEBRA申请中，我们提出了以下两个具体目标：目标1：在新型Ion Torrent PGMTM半导体测序仪上使用新型UV交联和RNA免疫沉淀以及高通量测序（CLIP-seq）方案，鉴定miR-495和HuD的mRNA靶标，并绘制其特异性结合位点。这些研究将使用体外和体内验证方法以及独特的生物信息学工具和新的竞争研究来定义HuD和miR-495如何能够以相反的方式调节相同的靶标。目标二：为了测试HuD与miR 495与其靶标的体内竞争性相互作用对动物对可卡因的动机（Aim 2a）和共享靶mRNA的转录后控制（Aim 2b）的功能后果。这些研究将使用HuD过表达小鼠和慢病毒构建体的立体定位注射来增加或减少核壳中的miR-495水平。我们认为我们的申请适合CEBRA计划，因为目标1中提出的同时鉴定HuD和miR-495的共同靶点和结合位点的尖端测序方法是高度创新的，并且具有潜在的高风险。我们的应用也是显着的意义和潜在的高影响，因为这些研究将揭示新的分子靶点和未探索的分子机制，涉及RNA结合蛋白和microRNA之间的竞争在药物成瘾。更好地了解这些调节机制是将这些新工具应用于成瘾研究并最终治疗这种疾病的先决条件。 公共卫生相关性：虽然转录后机制在基因表达的控制中起着至关重要的作用，但它们在成瘾行为建立中的作用却很少受到关注。因此，识别mR-495和HuD的分子靶点，这两种转录后调节因子与相同的靶序列相互作用，在基因表达和可卡因动机中具有相反的作用，将揭示药物成瘾期间突触可塑性适应不良变化的新机制，并提供潜在的新干预靶点。

DESCRIPTION (provided by applicant): Controlling motivation for cocaine is critical for the successful long-term treatment of cocaine addiction, which may require reversal of drug-induced changes in gene expression. Although post-transcriptional mechanisms play a vital role in the control of gene expression, their role in drug abuse has received little attention. RNA binding proteins and microRNAs serve as master switches controlling gene expression, with mRNA stability estimated to control about 20% of brain-expressed genes. Our research suggests that the RNA-binding protein HuD and the microRNA miR-495 play opposite roles in the control of addiction-related gene expression and behavior: 1) they are predicted to bind the same GU-rich sequence in mRNAs; 2) their binding sites are overrepresented in transcripts from an addiction-related gene (ARG) database; 3) they show differential regulation by cocaine, with miR-495 downregulated and HuD upregulated in the nucleus accumbens; 4) in vitro manipulations of these molecules result in opposite effects on the expression of two of their target genes, BDNF and arc; and 5) most importantly, in vivo manipulations of these molecules show contrasting effects on the motivation for cocaine. Based upon these results, we hypothesize that HuD and miR-495 compete for binding to the same sequences to control the expression of ARGs and motivation for drug in opposing directions. To test this hypothesis, in the CEBRA application we propose the following two specific aims: Aim 1: To identify mRNA targets of miR-495 and HuD and to map their specific binding sites using a novel UV- crosslinking and RNA immunoprecipitation and high throughput sequencing (CLIP-seq) protocol on the new Ion Torrent PGMTM semiconductor sequencers. These studies will use in vitro and in vivo validation methods as well as unique bioinformatics tools and novel competition studies to define how HuD and miR-495 are able to regulate the same targets in an opposite manner. Aim 2: To test the functional consequences of the competitive interactions of HuD vs. miR495 with their targets in vivo both on the motivation of animals for cocaine (Aim 2a) and the post-transcriptional control of shared target mRNAs (Aim 2b). These studies will use HuD overexpressor mice and stereotaxic injections of lentiviral constructs to increase or decrease miR-495 levels in the nucleus accumbens shell. We consider our application to be appropriate for the CEBRA initiative as the cutting-edge sequencing method proposed in Aim 1 to simultaneously identify the common targets and binding sites of HuD and miR-495 is highly innovative and potentially of high risk. Our application is also markedly significant and potentialy of high-impact as these studies will uncover novel molecular targets and unexplored molecular mechanisms involving the competition between RNA-binding proteins and microRNAs in drug addiction. A better understanding of these regulatory mechanisms is a pre-requisite for the application of these new tools in addiction research and ultimately in the treatment of this disorder. PUBLIC HEALTH RELEVANCE: Although post-transcriptional mechanisms play a vital role in the control of gene expression, their role in the establishment of addictive behaviors has received very little attention. Therefore, the identification of the molecular targets of mR-495 an HuD, two post-transcriptional regulators that interact with the same target sequences with opposing effects in gene expression and motivation for cocaine will uncover new mechanisms underlying the maladaptive changes in synaptic plasticity during drug addiction and provide potential new targets for intervention.