Cocaine Regulation of FosB Splicing

描述（由申请人提供）：尽管最近的研究已经揭示了大量关于药物成瘾的分子基础，但我们对与慢性药物摄入相关的基因表达变化的理解仍存在许多空白。在慢性可卡因暴露后发生的一个显著改变是转录因子d FosB的非常稳定的同种型在纹状体中的积累，所述转录因子d FosB由FosB转录物的选择性剪接产生。δ FosB亚型可能介导一些与药物成瘾有关的神经和行为改变。在delta FosB的选择性剪接中起作用的因素尚未确定。为了开始了解导致产生稳定的dFosB的转录后事件，我们将检验顺式和反式作用因子在这种调节中都很重要的假设。FosB的突变分析将用于鉴定前体mRNA剪接所必需的序列。将进行交联和RNA结合研究，以确定哪些反式作用因子与从组织培养细胞和脑中制备的剪接提取物中的RNA调节序列结合。这些研究中鉴定的因子将在剪接测定中进行测试，以确定它们是否在体外调节剪接。将使用原位杂交组织化学确定调节剪接因子在脑内的分布。将改变调节因子的表达以确定对体内FosB的影响。这些研究将确定在长期使用可卡因期间调节剪接所需的因素，并导致对所涉及机制的理解。由于dFosB也是由长期服用其他滥用药物，如安非他明，尼古丁和阿片类药物引起的，本研究获得的结果也可能导致更好地了解药物成瘾。

DESCRIPTION (provided by applicant): Although recent studies have revealed a great deal about the molecular basis of drug addiction, numerous gaps remain in our understanding of the changes in gene expression that are associated with chronic drug intake. One prominent alteration that occurs upon chronic cocaine exposure is accumulation in the striatum of very stable isoforms of the transcription factor d FosB that is produced by alternative splicing of the FosB transcript. The delta FosB isoforms may mediate some of the neural and behavioral modifications that occur with drug addiction. The factors that play a role in the alternative splicing of delta FosB have not yet been identified. To begin to understand the posttranscriptional events that lead to the production of stable dFosB we will test the hypothesis that both cis- and trans-acting factors are important in this regulation. Mutation analysis of FosB will be used to identify the sequences essential for pre-mRNA splicing. Cross-linking and RNA-binding studies will be undertaken to determine which trans-acting factors bind to the RNA regulatory sequences in splicing extracts prepared from tissue culture cells and brains. Factors identified in these studies will be tested in splicing assays to determine if they regulate splicing in vitro. The distribution of the regulatory splicing factors within the brain will be determined using in situ hybridization histochemistry. The expression of the regulatory factors will be changed in order to determine the effect on FosB in vivo. These studies will identify the factors required for the regulation of splicing during chronic cocaine use and lead to an understanding of the mechanisms involved. Because dFosB is also induced by chronic administration of other drugs of abuse, such as amphetamine, nicotine and opiates, the results obtained in this study may also lead to a better understanding of drug addiction.