Regulation of mRNA Transport in Human Cells

描述（由申请人提供）：信使（m）RNA从细胞核向细胞质的输出是真核基因表达途径中的重要步骤，并影响细胞生理学的各个方面。本项目的目标是通过破译两种必需蛋白质人（h）GlelB和hDbp 5的功能来阐明人mRNA输出的精确机制。利用芽殖酵母S.在酿酒酵母模型中，我们的实验室最近发现，与肌醇六磷酸（IP 6）结合的yGel是最大限度地刺激yDbpS的RNA依赖性ATP酶活性所必需的。yDbp 5转化为ADP结合形式，然后触发输出的信使核糖核蛋白颗粒（mRNP）的蛋白质组成的变化。我们假设，出口机制是保守的人类细胞，并在一个致命的人类运动神经元退行性疾病的扰动。为了分析人类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hGel B和hDbp 5的生化特性。这项工作将利用一组在我们实验室中建立良好的测定方法。将进行体外结合测定以测试IP 6与hGel B的相互作用。为了测试活化，将用hDbp 5、hGel B和IP进行ATP酶测定。由于hDbp 5潜在地充当RNA解旋酶和RNP重塑剂，因此将对hGel B和IP 6在这些功能中的作用进行直接测定。将进行与重组hGel B和hDbpS的体外蛋白质-蛋白质相互作用，以及来自人组织培养细胞的体内免疫共沉淀研究.这些测定的结果将为hGel B、IP 6和hDbpS在mRNA输出中的特异性功能提供证据，并可能鉴定对其他新型细胞辅因子的需求。在目的二中，将研究干扰hGel B-Finmajor突变的机制。该突变等位基因最近被报道为LCCS 1（致死性先天性收缩综合征1）的因果联系。将测定重组纯化的hGlel B-Finmajor蛋白的IP 6结合、hDbpS活化和结合。将使用人组织培养细胞中的瞬时表达实验来比较野生型和hGel 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-FinmajOR的表达替代，并且将进行mRNA输出功能的原位测定。这些研究将使人们深入了解疾病的机制。公共卫生相关性：mRNA输出是一个基本的细胞过程，对真核细胞的活力至关重要。这一过程的失调与病毒感染、癌症过程有关，现在可能与神经元发育有关。了解正常的输出机制将使这种疾病过程中的扰动能够被检测到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.