Regulation of mRNA Transport in Human Cells

描述（由申请人提供）：信使RNA从细胞核向细胞质的输出是真核生物基因表达途径的重要步骤，影响细胞生理的各个方面。该项目的目标是通过解析人类(h) GlelB和hDbp5两种必需蛋白的功能来阐明人类mRNA输出的确切机制。利用出芽酵母(y) S. cerevisiae模型，我们的实验室最近发现，yGlel与肌醇六磷酸（IP6）结合，是最大限度地刺激yDbpS的rna依赖性atp酶活性所必需的。yDbp5转化为adp结合形式触发输出信使核糖核蛋白颗粒（mRNP）蛋白质组成的变化。我们假设输出机制在人类细胞中是保守的，并在致命的人类运动神经元退行性疾病中受到干扰。为了分析人体细胞中的过程并验证这一假设，提出了两个目标。目的一，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建立的一系列测定方法。体外结合实验将检测IP6与hGlel B的相互作用。为了检测激活，ATPase实验将与hDbp5、hGlel B和IPs一起进行。由于hDbp5可能同时作为RNA解旋酶和RNP重塑酶，因此将对hDbp5 B和IP6在这些功能中的作用进行直接检测。将进行与重组hGlel B和hDbpS的体外蛋白-蛋白相互作用，以及人组织培养细胞的体内共免疫沉淀研究。这些试验的结果将为hgleb、IP6和hDbpS在mRNA输出中的特定功能提供证据，并可能确定对其他新型细胞辅助因子的需求。在目标二中，将研究干扰hgll B-Finmajor突变的机制。这个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。重组纯化的hGlel B-Finmajor蛋白将检测IP6结合、hDbpS激活和结合。在人组织培养细胞中的瞬时表达实验将用于比较野生型和hgll B-Finmajor蛋白的亚细胞定位。使用siRNA敲低策略，野生型hgll将被hgll b - finmaj0的表达所取代，并进行mRNA输出功能的原位检测。这些研究将有助于深入了解该病的发病机制。公共卫生相关性：mRNA输出是一个基本的细胞过程，对真核细胞的生存能力至关重要。这一过程的失调与病毒感染、癌症过程有关，现在也可能与神经元发育有关。了解正常的输出机制将使这些疾病过程中的扰动被发现并有针对性地进行治疗。

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.