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，信使核糖核酸的稳定性被认为控制了大约10%的人类基因。研究最多的顺式作用元件之一是存在于几个不稳定的mRNAs的3‘非翻译区(3’UTR)中的富AU元件(ARE)。这些序列是RNA结合蛋白的靶标，例如神经元特异性和可塑性相关的RNA结合蛋白HUD。我们最近发现了三个新的HUD结合基序，其中两个是富U的，因此被称为HUD-Ares。利用生物信息学分析，我们发现在mRNAs的3‘UTRs中存在HUD-ARE基序的过度表达，这些基序与成瘾机制有关，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的microRNAs的靶标。在BDNF和GAP-43的情况下，HUD识别的基序也包含miR-495的目标序列。鉴于RNA结合蛋白和microRNAs识别的序列之间的重叠以及这些位点在成瘾相关基因(ARGs)的3‘UTRs中的过度表达，我们认为RNA结合蛋白(如HUD)可以与microRNAs(如miR-495)竞争与物质滥用相关的基因的转录后控制。为了验证这一假说，我们建议：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HUD-Ares和毒品成瘾相关基因3‘UTR中的microRNA位点的患病率和共存位置；2)在体外实验中测试HUD和miR-495之间对BDNF和GAP-43 mRNAs稳定性的功能竞争，并在体内评估这些相互作用的意义。这项R03拨款中描述的实验是解决microRNAs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，这是一个新的和重要的调控过程。 公共卫生相关性：尽管转录后机制在基因表达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多的转录后调控元件；2)这些元件被两个关键的转录后调节元件--microRNAs和RNA结合蛋白识别；3)这些分子可能竞争控制基因的表达。这项R03拨款中描述的实验是解决microRNAs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，这是一个新的和重要的调控过程。

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.