Ethanol-induced NMDA R1 mRNA Stabilization

乙醇诱导 NMDA R1 mRNA 稳定

• 批准号: 8690257
• 负责人: MEENA KUMARI
• 金额: $ 3.36万
• 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 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8690257
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Acids; Alcohol dependence; Alcohols; Antibodies; Biological Assay; Brain; Cell Culture Techniques; Cell Nucleus; Cells; Chronic; Code; Complementary DNA; Computer software; Cytosol; Dactinomycin; Data; Development; Disease; Ethanol; Exons; Genes; Genetic Transcription; Goals; Grant; Half-Life; In Situ; In Situ Hybridization; In Vitro; Incubated; Lead; Length; Mediating; Memory; Messenger RNA; Methionine; Molecular; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; NR1 gene; Neurons; Oryctolagus cuniculus; Pharmacology; Physiology; Plasmids; Play; Process; Protein Biosynthesis; Proteins; RNA; RNA Interference; RNA Probes; RNA Splicing; RNA-Protein Interaction; Regulation; Reporter Genes; Research Personnel; Reticulocytes; Ribonucleases; Role; Scintillation Counting; Site; System; Testing; Transfection; Translations; Variant; Western Blotting; alcohol effect; alcohol exposure; alcoholism therapy; chronic alcohol ingestion; design; fetal; immunocytochemistry; in vivo; internal control; knock-down; mRNA Stability; neurotransmission; novel therapeutic intervention; polypeptide; programs; receptor; time interval; translation assay

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 NR1splice variant (NR1-4a) at the mRNA & polypeptide levels in cultured fetal cortical neurons (FCN) (Kumari, 2001) uggesting 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 (Anji et al., 2004). 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. -

NR1亚基是单基因的产物，是关键亚基，而功能性NMDA受体则不是 没有它就形成了。NMDA受体是乙醇的重要作用部位。慢性酒精暴露增加 在体内和体外调节NMDA受体的数量/功能，伴随着NR1和NR2B的增加 蛋白质水平。在四种NR1剪接变体中，慢性乙醇只支持一种NR1剪接的表达 培养的胎儿皮质神经元(FCN)中mRNA和多肽水平的突变体(NR1-4a)(Kumari，2001) 提示乙醇处理的神经元中NMDA受体的生理学和药理学改变。 此外，慢性乙醇介导的NR1mRNA半衰期的增加需要从头蛋白的参与 综合。进一步的分子检测发现，在NR13‘非编码区中有两个顺式作用区域。一个顺位- 作用区与三种反式作用蛋白相互作用，慢性乙醇上调一种反式作用蛋白 FCN中的蛋白质(Anji等人，2004年)。该项目的长期计划是阐明分子机制(S) 参与乙醇介导的FCN中NR1亚单位的调节。在这笔赠款中，提出了以下建议：(1) 描述机制(S)包括核内剪接改变NR1mRNA水平的剪接变异体 乙醇处理FCN；确定RNA-蛋白质相互作用对NR1mRNA的重要性(2)稳定性；(3) 运输与本地化；(4)翻译。这些目标将通过(A)确定#年的半衰期来实现。 NR1剪接变异体和FCN中外显子5的剪接； 瞬时转染法或稳定转染法确定顺式和反式因子对NR1mRNA稳定性的影响 NR1mRNA转运(使用放线菌素-D半衰期研究)；转运和定位(通过原位杂交和 免疫细胞化学)；和翻译(使用体外翻译系统)。一个更彻底的 了解乙醇调节R1mRNA的相关分子机制 稳定性/翻译可能允许设计与酒精相关的疾病的新治疗方法。 NMDA受体介导神经传递，在记忆形成中发挥重要作用。 这些受体也会受到长期饮酒的影响，并在酒精的形成中发挥作用。 依赖。更好地了解酒精对大脑中NMDA受体调节的影响将 使酒精中毒得到更好的治疗。-

项目成果

Expression of α-subunit of α-glucosidase II in adult mouse brain regions and selected organs.

α-葡萄糖苷酶 II 的 α 亚基在成年小鼠大脑区域和选定器官中的表达。

• DOI: 10.1002/jnr.23470
• 发表时间: 2015
• 期刊: Journal of neuroscience research
• 影响因子: 4.2
• 作者: Anji,Antje;Miller,Hayley;Raman,Chandrasekar;Phillips,Mathew;Ciment,Gary;Kumari,Meena
• 通讯作者: Kumari,Meena

A cis-acting region in the N-methyl-d-aspartate R1 3'-untranslated region interacts with the novel RNA-binding proteins beta subunit of alpha glucosidase II and annexin A2--effect of chronic ethanol exposure in vivo.

• DOI: 10.1111/j.1460-9568.2011.07857.x
• 发表时间: 2011-10
• 期刊: The European journal of neuroscience
• 作者: Anji A;Kumari M
• 通讯作者: Kumari M

Guardian of Genetic Messenger-RNA-Binding Proteins.

• DOI: 10.3390/biom6010004
• 发表时间: 2016-01-06
• 期刊: Biomolecules
• 影响因子: 5.5
• 作者: Anji A;Kumari M
• 通讯作者: Kumari M