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）：信使（m）RNA从细胞核输出到细胞质是真核基因表达途径中的重要步骤，并且影响细胞生理学的各个方面。该项目的目标是通过破译人类 (h) Gle1B 和 hDbp5 两种必需蛋白的功能，阐明人类 mRNA 输出的精确机制。使用芽殖酵母 (y) 酿酒酵母模型，我们的实验室最近发现，yGlel 与六磷酸肌醇 (IP6) 结合，需要最大限度地刺激 yDbpS 的 RNA 依赖性 ATP 酶活性。 yDbp5 转化为 ADP 结合形式会触发输出的信使核糖核蛋白颗粒 (mRNP) 的蛋白质组成发生变化。我们假设输出机制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。为了分析人类细胞的过程并检验这一假设，提出了两个目标。在目标一中，将分析重组纯化的 hGleB 和 hDbp5 的生化特性。这项工作将利用我们实验室成熟的一系列检测方法。将进行体外结合测定以测试 IP6 与 hGlel B 的相互作用。为了测试激活情况，将使用 hDbp5、hGlel B 和 IP 进行 ATPase 测定。由于 hDbp5 可能同时充当 RNA 解旋酶和 RNP 重塑剂，因此将针对 hGle1 B 和 IP6 在这些功能中的作用进行直接测定。将进行与重组 hGleB 和 hDbpS 的体外蛋白质-蛋白质相互作用，以及来自人体组织培养细胞的体内共免疫沉淀研究。这些测定的结果将为 hGleB、IP6 和 hDbpS 在 mRNA 输出中的特定功能提供证据，并可能确定其他新型细胞辅助因子的需求。在目标二中，将研究 hGlel B-Finmajor 突变受到干扰的机制。该突变等位基因最近被报道为 LCCS1（致死性先天性收缩综合征 1）的因果关系。将检测重组纯化的 hGlel B-Finmajor 蛋白的 IP6 结合、hDbpS 激活和结合。人体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，野生型 hGlel 将被 hGlel B-Finmaj0r 的表达取代，并将进行 mRNA 输出功能的原位测定。这些研究将深入了解疾病机制。公共健康相关性：mRNA 输出是一个基本的细胞过程，对于真核细胞的生存至关重要。这一过程的失调与病毒感染、癌症过程有关，现在还可能与神经元发育有关。了解正常的输出机制将有助于检测此类疾病过程中的扰动并进行针对性治疗。