Regulation of mRNA Transport in Human Cells

人体细胞中 mRNA 运输的调节

• 批准号: 8078112
• 负责人: Kristen Nicole Noble
• 金额: $ 3.68万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8078112
• 关键词: ATP phosphohydrolase; Alleles; Amino Acids; Area; Basic Science; Binding; Biochemical; Biological Assay; Cell Cycle; Cell Line; Cell Nucleus; Cell physiology; Cells; Clinical Skills; Co-Immunoprecipitations; Contracture; Cultured Cells; Cytoplasm; Defect; Degenerative Disorder; Development; Disease; Eukaryotic Cell; Fetal Development; Functional disorder; Gene Expression; Goals; Human; In Situ; In Vitro; Inherited; Laboratories; Link; Malignant Neoplasms; Messenger RNA; Modeling; Molecular; Motor Neurons; Mutation; Nuclear Envelope; Nuclear Pore Complex; Pathway interactions; Phytic Acid; Process; Property; Proteins; RNA; RNA Helicase; RNA-dependent ATPase; Recombinants; Regulation; Reporting; Research; Research Training; Role; Saccharomyces cerevisiae; Saccharomycetales; Signal Transduction; Small Interfering RNA; Syndrome; Testing; Training; Virus Diseases; Work; cell growth; extracellular; fetal; human disease; human tissue; in vivo; insight; mRNA Export; messenger ribonucleoprotein; mutant; neuron development; novel; particle; programs; protein protein interaction; public health relevance; research study; response; tissue culture

DESCRIPTION (provided by applicant): The export of messenger (m)RNA from the nucleus to the cytoplasm is an essential step in the eukaryotic gene expression pathway and impacts all aspects of cell physiology. The goal of this project is to elucidate the precise mechanism of human mRNA export by deciphering the function of two essential proteins, human (h) GlelB and hDbp5. Using the budding yeast (y) S. cerevisiae model, our laboratory recently found that yGlel, bound to inositol hexakisphosphate (IP6), is required to maximally stimulate the RNA-dependent ATPase activity of yDbpS. Conversion of yDbp5 to the ADP-bound form then triggers changes in the protein composition of the exported messenger ribonucleoprotein particle (mRNP). We hypothesize that the mechanism of export is conserved in human cells, and perturbed in a lethal human motoneuron degenerative disease. To analyze the process in human cells and test this hypothesis, two aims are proposed. In aim one, the biochemical properties of recombinant purified hGlel B and hDbp5 will be analyzed. This work will utilize a battery of assays that are well established in our laboratory. In vitro binding assays will be performed to test for IP6 interaction with hGlel B. To test for activation, ATPase assays will be performed with hDbp5, hGlel B and IPs. As hDbp5 potentially acts as both an RNA helicase and an RNP remodeler, direct assays will be conducted for the role of hGlel B and IP6 in these functions. In vitro protein- protein interactions with recombinant hGlel B and hDbpS will be conducted, as well as in vivo co- immunoprecipitation studies from human tissue culture cells. Results of these assays will provide evidence for the specific functions of hGlel B, IP6, and hDbpS in mRNA export, and potentially identify requirements for other novel cellular co-factors. In aim two, the mechanism by which the hGlel B-Finmajor mutation is perturbed will be investigated. This mutant allele was recently reported as the causal link in LCCS1 (lethal congenital contractile syndrome 1). Recombinant purified hGlel B-Finmajor protein will be assayed for IP6 binding, hDbpS activation and binding. Transient expression experiments in human tissue culture cells will be used to compare the subcellular localizations of wild-type and hGlel B-Finmajor proteins. Using siRNA knockdown strategies, wild-type hGlel will be replaced by expression of hGlel B-Finmaj0r and in situ assays for mRNA export function will be conducted. These studies will give insight into the disease mechanism. Public Health Relevance: mRNA export is a basic cellular process that is essential to the viability of eukaryotic cells. Dysregulation of this process is implicated in viral infection, cancer processes, and now potentially in neuronal development. Understanding the normal export mechanism will allow perturbations in such disease processes to be detected and targeted for treatment.

描述（由申请人提供）：从核从细胞核到细胞质的导出是真核基因表达途径的重要步骤，并影响细胞生理的各个方面。该项目的目的是通过破译两个必需蛋白质人（H）Glelb和HDBP5的功能来阐明人mRNA导出的精确机制。使用萌芽的酵母菌（Y）S。酿酒酵母模型，我们的实验室最近发现，与肌醇六磷酸（IP6）结合的YGLEL是需要最大程度地刺激YDBPS的RNA依赖性ATPase活性的。将YDBP5转换为ADP结合形式，然后触发导出的核核糖蛋白颗粒（MRNP）的蛋白质组成的变化。我们假设出口机理在人类细胞中是保守的，并且在致命的人类运动神经元退行性疾病中受到干扰。为了分析人类细胞中的过程并检验该假设，提出了两个目标。在目标中，将分析重组纯化的HGLEL B和HDBP5的生化特性。这项工作将利用我们实验室中确定的一系列测定法。将进行体外结合测定，以测试与HGLELB的IP6相互作用。为了测试激活，将使用HDBP5，HGLEL B和IPS进行ATPase分析。由于HDBP5可能充当RNA解旋酶和RNP重塑，因此将直接测定HGLEL B和IP6在这些功能中的作用。将进行体外蛋白质蛋白与重组HGLEL B和HDBPS的相互作用，以及来自人类组织培养细胞的体内共沉淀研究。这些测定结果的结果将为mRNA导出中HGLEL B，IP6和HDBP的特定功能提供证据，并有可能确定对其他新型细胞co骨因子的要求。在目标两个方面，将研究HGLEL B-FINMAJOR突变的机制。该突变等位基因最近被报道为LCCS1的因果关系（致命的先天性收缩综合征1）。重组纯化的HGLEL B-FINMAJOR蛋白将用于IP6结合，HDBPS激活和结合。人类组织培养细胞中的瞬时表达实验将用于比较野生型和HGLEL B-FINMAJOR蛋白的亚细胞定位。使用siRNA敲低策略，将通过HGLEL B-FINMAJ0R的表达代替野生型HGLEL，并将进行原位MRNA输出功能。这些研究将深入了解疾病机制。公共卫生相关性：mRNA输出是一种基本的细胞过程，对真核细胞的生存能力至关重要。该过程的失调与病毒感染，癌症过程有关，现在可能与神经元发育有关。了解正常的出口机制将允许在这种疾病过程中扰动并针对治疗。