Role of MicroRNAs and RNA-Binding Proteins in Addiction-Related Gene Expression

描述（由申请人提供）：尽管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。其中，mRNA的稳定性估计控制着所有人类基因的约10%。研究最多的顺式作用元件之一是存在于几种不稳定mRNA的3 '非翻译区（3' UTR）中的富含AU的元件（ARE）。这些序列是RNA结合蛋白如神经元特异性和可塑性相关的RNA结合蛋白HuD的靶标。我们最近确定了三个新的结合图案的HUD，其中两个是U-丰富，因此被称为HuD-ARE。使用生物信息学分析，我们发现在与成瘾机制相关的mRNA的3'UTR中存在HuD-ARE基序的过度表达，包括BDNF和GAP-43。ARE序列不仅是RNA结合蛋白的靶标，而且最近被证明是特定microRNA的靶标。在BDNF和GAP-43的情况下，HuD识别的相同ARE基序也包含miR-495的靶序列。考虑到RNA结合蛋白和microRNA识别的序列之间的重叠以及这些位点在成瘾相关基因（ARG）的3'UTR中的过度表达，我们提出RNA结合蛋白（如HuD）可以与microRNA（如miR-495）竞争与药物滥用相关的基因的转录后控制。为了验证这一假设，我们提出：1）使用生物信息学工具从公共领域数据库和我们自己的微阵列研究中挖掘数据，以调查与药物成瘾相关的基因的3 'UTR中HuD-ARE和microRNA位点的流行和共定位，以及2）实验测试HuD和miR-495之间对BDNF和GAP-495稳定性的功能竞争。43种mRNA的相互作用，并在可卡因自我给药的动物模型中评估这些相互作用的体内意义。R 03资助中描述的实验是解决microRNA和RNA结合蛋白在控制成瘾相关基因表达中可能相互作用的第一步，这是一种新的重要调控过程。 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。我们的初步结果表明：1）与药物成瘾相关的基因具有异常高数量的转录后调控元件，2）这些元件被两个关键的转录后调控因子microRNA和RNA结合蛋白识别，3）这些分子可以竞争控制基因表达。R 03资助中描述的实验是解决microRNA和RNA结合蛋白在控制成瘾相关基因表达中可能相互作用的第一步，这是一种新的重要调控过程。

DESCRIPTION (provided by applicant): 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. Amongst these, mRNA stability is estimated to control about 10% of all human genes. One of the most studied cis-acting elements is the AU-rich element (ARE) present in the 3'untranslated region (3'UTR) of several unstable mRNAs. These sequences are targets of RNA-binding proteins such as the neuronal-specific and plasticity-associated RNA-binding protein HuD. We have recently identified three new binding motifs for HuD; two of which are U-rich and thus termed HuD-AREs. Using bioinformatics analyses, we found that there is an overrepresentation of HuD-ARE motifs in the 3' UTRs of mRNAs that are associated with mechanisms of addiction, including BDNF and GAP-43. ARE sequences are not only targets of RNA-binding proteins but also they were recently shown to be targets of specific microRNAs. In the case of BDNF and GAP-43, the same ARE motifs that are recognized by HuD also contain target sequences for miR-495. Given that the overlap between sequences recognized by RNA-binding proteins and microRNAs and the overrepresentation of these sites in the 3' UTRs of addiction-related genes (ARGs), we propose that RNA-binding proteins such as HuD could compete with microRNAs such as miR-495 for the post-transcriptional control of genes associated with substance abuse. To test this hypothesis we propose: 1) To use bioinformatics tools to mine data from public domain databases and from our own microarray studies to investigate the prevalence and co-localization of HuD-AREs and microRNA sites in the 3'UTR of genes associated with drug addiction and 2) To experimentally test the functional competition between HuD and miR-495 on the stability of the BDNF and GAP-43 mRNAs in vitro and to assess the significance of these interactions in vivo in an animal model of cocaine-self-administration. The experiments described in this R03 grant are the first steps to address the possible interactions of microRNAs and RNA-binding proteins in the control of addiction-related gene expression, a novel and important regulatory process. 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. Our preliminary results indicate that 1) genes associated with drug addiction have an unusual high number of post-transcriptional regulatory elements, 2) these elements are recognized by two key post-transcriptional regulators, microRNAs and RNA-binding proteins and 3) these molecules could compete to control gene expression. The experiments described in this R03 grant are the first steps to address the possible interactions of microRNAs and RNA-binding proteins in the control of addiction-related gene expression, a novel and important regulatory process.