ETHANOL INDUCED NMDA R1 MRNA STABILIZATION

乙醇诱导 NMDA R1 mRNA 稳定

• 批准号: 6610947
• 负责人: MEENA KUMARI
• 金额: $ 11.58万
• 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 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6610947
• 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，是乙醇作用的重要部位。 在体内和体外慢性乙醇处理后， 数量和功能上调，伴随着R1 和R2 B多肽水平。 体外相似乙醇处理 将R1 mRNA的半衰期从16小时增加到24小时以上（Kumari和 Ticku，1998 a），表明转录后机制起作用 增加皮层神经元NMDA受体的数量， 乙醇处理（50 mM，5天）。 最近，我们观察到， 新的蛋白质合成是乙醇诱导的稳定所必需的， R1 mRNA（Kumari和Ticku，1998 b），表明乙醇诱导的 未知的蛋白质因子介导这种效应。 长期计划 项目是阐明转录后机制参与 在胎儿皮层神经元NMDA R1 mRNA的稳定中， 慢性酒精治疗。 本提案中有待检验的假设 是（1）鉴定特定的RNA序列（或顺式作用调控序列）， R1 mRNA的元件）;和（2）乙醇诱导的 细胞质蛋白（或反式作用因子），与顺式 作用RNA调节序列。 这些目标将通过以下方式实现： (a)检测乙醇是否诱导稳定的R1 剪接变体;（B）描绘顺式作用调节区 在R1 mRNA的一级序列内使用无细胞mRNA衰变 分析和细胞转染;（c）剖析顺式作用序列 在上面定义的调控区域内，使用由 与RNA偶联的巢式缺失、接头扫描和碱基取代 凝胶迁移试验和细胞转染，最后（d）鉴定 反式作用因子的性质（通过UV交联和西北 分析. 更深入地了解相关分子 乙醇调节NMDA R1 mRNA稳定性的机制可能 允许设计新的治疗方法来治疗酒精相关的 疾病