ETHANOL INDUCED NMDA R1 MRNA STABILIZATION

乙醇诱导 NMDA R1 mRNA 稳定

• 批准号: 6497160
• 负责人: MEENA KUMARI
• 金额: $ 3.44万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 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 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6497160
• 关键词: NMDA receptors; chemical stability; ethanol; gel mobility shift assay; gene induction /repression; genetic regulatory element; laboratory mouse; messenger RNA; neuropharmacology; northern blottings; pharmacogenetics; posttranscriptional RNA processing; protein structure function; tissue /cell culture; transcription factor; western blottings

The N-methyl-D-aspartate (NMDA) receptor, an excitatory neurotransmitter receptor in the brain, is an important site of action of ethanol. Following chronic ethanol treatment in vivo and in vitro, NMDA receptor number and function are upregulated, with a concomitant increase in R1 and R2B polypeptide levels in vitro. Similar ethanol treatment in vitro increases R1 mRNA half-life from 16 h to more than 24 h (Kumari and Ticku, 1998a) indicating that post-transcriptional mechanisms operate to augment NMDA receptor number in cortical neurons exposed to chronic ethanol treatment (50 mM, 5 days). More recently, we observed that de novo protein synthesis is required for ethanol-induced stabilization of R1 mRNA (Kumari and Ticku, 1998b), suggesting that ethanol-induced unknown protein factor(s) mediate this effect. Long term plans of this project are to elucidate the post-transcriptional mechanisms involved in the stabilization of NMDA R1 mRNA in fetal cortical neurons exposed to chronic ethanol treatment. Hypothesis to be tested in this proposal are (1) to identify specific RNA sequences (or cis-acting regulatory elements) of the R1 mRNA; and, (2) the nature of ethanol-induced cytoplasmic protein(s) (or trans-acting factors) that interact with cis- acting RNA regulatory sequences. These objectives will be achieved by (a) examining whether ethanol induces transcription of a stable R1 splice-variant; (b) delineating the cis -acting regulatory region(s) within the primary sequence of the R1 mRNA using cell-free mRNA decay assay and cell transfections; (c) dissecting the cis-acting sequences within the regulatory region defined above using mutants created by nested deletion, linker scanning and base substitution coupled to RNA gel shift assays and cell transfections, and finally (d) identifying the nature of trans-acting factor(s) by UV cross-linking and Northwestern analysis. A more thorough understanding of the pertinent molecular mechanisms through which ethanol modulates NMDA R1 mRNA stability may permit the design of novel therapeutic approaches to alcohol-related diseases.

N-甲基-D-天冬氨酸 (NMDA) 受体，一种兴奋性神经递质 大脑中的受体，是乙醇的重要作用部位。 体内和体外长期乙醇治疗后，NMDA 受体 数量和功能上调，R1 随之增加 和R2B多肽体外水平。 类似的体外乙醇处理 将 R1 mRNA 半衰期从 16 小时延长至 24 小时以上（Kumari 和 Ticku, 1998a) 表明转录后机制起作用 增加暴露于慢性暴露的皮质神经元中的 NMDA 受体数量 乙醇处理（50 mM，5 天）。 最近，我们观察到 乙醇诱导的稳定需要 novo 蛋白质合成 R1 mRNA（Kumari 和 Ticku，1998b），表明乙醇诱导 未知的蛋白质因子介导这种效应。 本次的长期计划 项目旨在阐明所涉及的转录后机制 暴露的胎儿皮质神经元中 NMDA R1 mRNA 的稳定性 慢性乙醇治疗。 本提案中待检验的假设 (1) 识别特定的RNA序列（或顺式作用调节序列） R1 mRNA的元件)； (2) 乙醇诱导的性质 与顺式相互作用的细胞质蛋白（或反式作用因子） 作用RNA调控序列。 这些目标将通过以下方式实现 (a) 检查乙醇是否诱导稳定 R1 的转录 剪接变体； (b) 划定顺式作用监管区域 使用无细胞 mRNA 衰减在 R1 mRNA 的一级序列中 测定和细胞转染； (c) 剖析顺式作用序列 在上面定义的调节区域内使用由以下方法创建的突变体 与 RNA 偶联的嵌套删除、接头扫描和碱基替换 凝胶迁移分析和细胞转染，最后 (d) 鉴定 通过 UV 交联和 Northwestern 分析反式作用因子的性质 分析。 对相关分子有更深入的了解 乙醇调节 NMDA R1 mRNA 稳定性的机制可能 允许设计针对酒精相关的新治疗方法 疾病。