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种不稳定mrna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（如神经元特异性和可塑性相关rna结合蛋白HuD）的靶标。我们最近为HuD确定了三个新的绑定基序；其中两个富含铀，因此被称为HuD-AREs。通过生物信息学分析，我们发现与成瘾机制相关的mrna（包括BDNF和GAP-43）的3' utr中存在过多的HuD-ARE基序。ARE序列不仅是rna结合蛋白的靶标，最近也被证明是特定microrna的靶标。在BDNF和GAP-43的情况下，HuD识别的相同的ARE基序也包含miR-495的靶序列。考虑到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' utr中的过度代表，我们提出，rna结合蛋白如HuD可以与microRNAs如miR-495竞争与药物滥用相关基因的转录后控制。为了验证这一假设，我们提出：1)使用生物信息学工具来挖掘数据从公共领域数据库和我们自己的微阵列研究调查的患病率和co-localization HuD-AREs和microRNA网站3 'utr基因与药物成瘾和2)实验测试功能之间的竞争住房和城市发展部和mir - 495 BDNF的稳定性和GAP-43 mrna在体外和体内评估这些交互的重要性在cocaine-self-administration动物模型。在这个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的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.