Ethanol-induced NMDA R1 mRNA Stabilization

乙醇诱导 NMDA R1 mRNA 稳定

• 批准号: 7038629
• 负责人: MEENA KUMARI
• 金额: $ 30.47万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7038629
• 关键词: NMDA receptors; RNA interference; RNA splicing; chemical stability; embryo /fetus tissue /cell culture; ethanol; fusion gene; gene induction /repression; genetic regulatory element; genetic translation; immunocytochemistry; in situ hybridization; intermolecular interaction; laboratory mouse; messenger RNA; neurons; neuropharmacology; nucleic acid quantitation /detection; pharmacogenetics; protein structure; protein structure function; receptor expression; regulatory gene; transcription factor; transfection

DESCRIPTION (provided by applicant): The NR1 subunit, a product of a single gene, is the key subunit as functional NMDA receptors are not formed without it. NMDA receptors are an important site of action of ethanol. Chronic ethanol exposure up regulates NMDA receptor number/function in vivo and in vitro with a concomitant increase in NR1 and NR2B protein levels. Out of four NR1 splice variants, chronic ethanol favors the expression of only one NR1 splice variant (NR1-4a) at the mRNA & polypeptide levels in cultured fetal cortical neurons (FCN) suggesting an altered physiology and pharmacology of NMDA receptors in ethanol treated neurons. Furthermore, chronic ethanol-mediated increase in NR1 mRNA half-life in vitro requires de novo protein synthesis. A closer molecular examination identified two cis-acting regions in the NR1 3' UTR. One cis- acting region interacts with three trans-acting proteins and chronic ethanol up regulates one trans-acting protein in FCN. Long term plan of this project is to elucidate molecular mechanism(s) involved in ethanol-mediated regulation of NR1 subunit in FCN. In this grant, the following are proposed: (1) delineate mechanism(s) including splicing in the nucleus that alter mRNA levels of NR1 splice variants in ethanol treated FCN; determine the importance of RNA-protein interactions on NR1 mRNA (2) stability; (3) transport and localization; and (4) translation. These goals will be achieved by (a) determining half-lives of NR1 splice variants and splicing of exon 5 in FCN; (b) generating & testing chimeric plasmids, by RNAi, transient transfection or stable transfection to determine role of cis and trans factors on NR1 mRNA stability, NR1 mRNA transport (half-life study using actinomycin-D); transport and localization (by in situ hybridiztion & immunocytochemistry); and translation (using an in vitro translation system). A more thorough understanding of the pertinent molecular mechanisms through which ethanol modulates R1 mRNA stability/translation may permit the design of novel therapeutic approaches to alcohol-related diseases. NMDA receptors mediate neurotransmission and play important roles in memory formation. These receptors are also effected by a chronic alcohol consumption and play a role in development alcohol dependence. A better understanding of the effects of alcohol on NMDA receptors regulation in the brain will lead to better treatment for alcoholism.

描述（由申请人提供）：NR1亚基是单个基因的产物，是关键亚基，因为没有它就无法形成功能性NMDA受体。NMDA受体是乙醇作用的重要部位。在体内和体外，慢性乙醇暴露上调NMDA受体数量/功能，同时NR1和NR2B蛋白水平升高。在四种NR1剪接变体中，在培养的胎儿皮质神经元（FCN）中，慢性乙醇只支持一种NR1剪接变体（NR1-4a）在mRNA和多肽水平上的表达，这表明乙醇处理的神经元中NMDA受体的生理和药理学发生了改变。此外，慢性乙醇介导的nr1mrna半衰期增加需要重新合成蛋白质。进一步的分子检查发现nr13 ' UTR中有两个顺式作用区域。一个顺式作用区与三个反式作用蛋白相互作用，慢性乙醇上调FCN中的一个反式作用蛋白。本项目的长期计划是阐明乙醇介导的FCN中NR1亚基调控的分子机制。在这项资助中，提出了以下建议：(1)描述包括细胞核剪接在内的机制，该机制改变了乙醇处理的FCN中NR1剪接变异体的mRNA水平；确定rna -蛋白相互作用对nr1mrna稳定性的重要性(2)；(3)运输和定位；(4)翻译。这些目标将通过(a)确定NR1剪接变异体的半衰期和FCN中外显子5的剪接来实现；(b)通过RNAi、瞬时转染或稳定转染制备和测试嵌合质粒，以确定顺式和反式因子对NR1 mRNA稳定性和NR1 mRNA转运的作用（使用放线菌素- d进行半衰期研究）；运输和定位（通过原位杂交和免疫细胞化学）；和翻译（使用体外翻译系统）。更深入地了解乙醇调节R1 mRNA稳定性/翻译的相关分子机制，可能有助于设计治疗酒精相关疾病的新方法。NMDA受体介导神经传递，在记忆形成中起重要作用。这些受体也受到长期饮酒的影响，并在酒精依赖的发展中发挥作用。更好地了解酒精对大脑中NMDA受体调节的影响将有助于更好地治疗酒精中毒。