Function and Regulation of a DEAD-box protein in mRNA Export

项目总结 信使核糖核酸的输出是真核生物基因表达程序的一个基本组成部分，它与 与上游(例如转录)和下游(例如RNA监测和翻译)事件相关联。 信使核糖核酸输出模型的发展及对输出在调控基因中作用的认识 表达(例如，选择性转录本的偏向输出)需要对信使核糖核酸输出进行定量评估， 包括动力学和分子机制。考虑到已有的知识，追求这一目标是及时的 积累和开发的技术(例如RNA适体标记系统)，对于了解 疾病发生在对这一细胞过程的扰动的背景下。要考虑的一个关键因素是Dbp5p，a DEAD-box蛋白(DBP)，通过调节RNA-蛋白质相互作用在出口中发挥关键作用 通过核孔复合体(NPC)定向运输mRNA。Dbp5p被激活(即增强 ATPase活性)由NPC组分Gle1p和Nup159p组成，尽管这些调节因子的影响 已经在体外(如稳态动力学和结构生物学)、动力学和热力学方面进行了研究 这一调控的起源，以及ATPase循环如何影响mRNA输出的空间和时间方面 VIVO，仍未解决。因此，迈向准确的信使核糖核酸输出模型的必要步骤是 定量了解(1)Dbp5p对ATP的利用和(2)Dbp5p对mRNA输出的影响 体内动力学，包括这些事件如何与NPC调节因子的相互作用相耦合。 具体地说，关于决定Dbp5p ATPase活性和相互作用的速率和平衡常数的知识 Nup159p和Gle1p是开发可组合的预测性NPC传输模型所必需的 体内测定信使核糖核酸的输出动力学和效率。目标1重点中的拟议研究工作 关于如何确定暂态(稳态前)动力学和定量平衡方法和分析 Nup159p和Gle1p与Dbp5p相互作用和调节。AIMS 2和AIMS 3使用单一的 解决信使核糖核酸输出的空间和时间方面的粒子核糖核酸成像和转录切割方法， 包括Dbp5p、Gle1p和Nup159p在调节出口中的作用。为了实现这些目标，新的 适用于其他DBPs和RNA传输系统的实验和分析方法将是 发展起来的。拟议活动的完成将建立一个量化的分子框架， 描述了调节因子如何作用于调节Dbp5p ATPase活性和mRNA转运，这是 基因表达、细胞生理学和疾病病理学的基础。 好了！

PROJECT SUMMARY mRNA export is a fundamental component of the gene expression program in eukaryotes, which is tightly linked to upstream (e.g. transcription) and downstream (e.g. RNA surveillance and translation) events. Development of mRNA export models and an understanding of the role export plays in regulated gene expression (e.g. biased export of select transcripts) requires a quantitative assessment of mRNA export that encompasses kinetics and molecular mechanism. Pursuing this goal is timely given the knowledge that has amassed and technology developed (e.g. RNA-aptamer tagging systems) and is vital to understanding how disease arises in the context of perturbations to this cellular process. A key factor to consider is Dbp5p, a DEAD-box protein (DBP) that plays a critical role in export by modulating RNA-protein interactions important to directional transport of mRNAs through nuclear pore complexes (NPCs). Dbp5p is activated (i.e. enhanced ATPase activity) by the NPC components Gle1p and Nup159p, and although the impact of these regulators have been studied in vitro (e.g. steady-state kinetics and structural biology), the kinetic and thermodynamic origins of this regulation, and how ATPase cycling influences spatial and temporal aspects of mRNA export in vivo, remain unresolved. Therefore, a necessary step towards accurate models of mRNA export are a quantitative understanding of (1) ATP utilization by Dbp5p and (2) the impact of Dbp5p on mRNA export dynamics in vivo, including how these events are coupled to interactions with NPC regulatory factors. Specifically, knowledge of the rate and equilibrium constants dictating Dbp5p ATPase activity and interactions with Nup159p and Gle1p is necessary for developing predictive NPC transport models that can be combined with measurements of mRNA export kinetics and efficiency in vivo. Proposed research efforts in Aim 1 focus on transient (pre-steady state) kinetic and quantitative equilibrium methods and analyses to determine how Nup159p and Gle1p interact with and regulate Dbp5p. Aims 2 and 3 employ in vivo studies using single particle RNA imaging and transcriptomic approaches to address spatial and temporal aspects of mRNA export, including the role of Dbp5p, Gle1p and Nup159p in modulating export. To accomplish these aims, new experimental and analysis methods readily applicable to other DBPs and RNA transport systems will be developed. Completion of the proposed activities will establish a quantitative molecular framework that describes how regulatory factors function to modulate Dbp5p ATPase activity and mRNA transport, which is fundamental to gene expression, cell physiology, and disease pathology. !