The role of XPO1 in nuclear export of RNA

XPO1 在 RNA 核输出中的作用

• 批准号: 10714250
• 负责人: Justin Taylor
• 金额: $ 38.38万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10714250
• 关键词: Address; Affect; Apoptosis; Binding; Binding Proteins; Biochemical; Biology; Cell Line; Cell Nucleus; Cells; Cytoplasm; Data; Disease; Eukaryotic Cell; Exposure to; Future; Gene Expression; Genetic Engineering; Genomics; Goals; Grant; Knock-in Mouse; Knowledge; Laboratories; Mediating; Medical; Messenger RNA; Mission; Mitotic Cell Cycle; Modeling; Molecular; Molecular Biology; Mutation; National Institute of General Medical Sciences; Nuclear; Nuclear Export; Pathogenesis; Pharmaceutical Preparations; Process; Protein Export Pathway; Proteins; Proteomics; RNA; RNA Splicing; Regulation; Research; Ribosomes; Role; Science; Signal Transduction; Translations; Work; exportin 1 protein; genetically modified cells; inhibitor; mRNA Translation; macromolecule; mouse model; mutant; nuclear factors of activated T-cells; overexpression; therapy design

Project Summary The focus of research in the Taylor laboratory is on nuclear export in eukaryotic cells, which we study by perturbing the normal function of the main exporter of proteins, XPO1 or Exportin-1. We use molecular biology, genomics, proteomics, and mouse modeling to determine the mechanisms of XPO1-mediated nuclear export function and the role of XPO1 in disease pathogenesis. We also study how XPO1 interacts with other proteins and molecules such as ribonucleic acid (RNA). In the next five years, the goal of the lab is to define the role of XPO1 in the nuclear export of RNA. The laboratory is currently pursuing the following projects. Defining the molecular effects of wildtype and mutant XPO1 on gene expression, splicing, and translation. Given the known role of XPO1 in the export of small nuclear RNA (snRNA) and ribosomal RNA (rRNA) via the binding of RNA binding proteins, we hypothesize that alterations in XPO1 may impact RNA splicing and/or mRNA translation. We have generated several genetically engineered models to allow endogenous expression of the XPO1 E571K or R749Q mutation, including conditional knockin mice. We have demonstrated through a variety of proteomic, biochemical, structural, and molecular studies that XPO1 E571K affects recognition of its cargo’s nuclear export signal (NES) and results specifically in altered export of of NFκB and NFAT transcription factor proteins. The aim of my first project is to continue to study the extent of how the XPO1 E571K alters nuclear export and to explore whether RNA export, and consequent mRNA splicing and translation, is affected through the mislocalization of RNA binding proteins. We have recently discovered another mutation, XPO1 R749Q, that affects nuclear export by increasing the export of proteins out of the nucleus. We plan to study which proteins this affects and how this affects cell growth, cell cycle and other homeostatic processes. We are also modeling overexpression of XPO1 and will compare how this overactive mutant compares to overexpression in relation to protein and RNA export. Drugs that selectively inhibit XPO1 will allow us to isolate the effects of XPO1- dependent export. Furthermore, our preliminary data show that cells with abnormal splicing due to SF3B1 mutations undergo apoptosis upon exposure to XPO1 inhibitors. We will also investigate the effects of XPO1 inhibition in mutant Sf3b1 expressing cells using genetically engineered cell lines and an Sf3b1 knockin mouse model. We hypothesis that XPO1 inhibition perturbs RNA export and affects gene expression and mRNA splicing given the known role of XPO1 to export small nuclear RNA via RNA-binding proteins. This work will serve as foundational data for future grant submissions investigating the biology of XPO1 and its role in nuclear export of RNA. The overarching goal of this research is aligned with the NIGMS’ mission to support new basic discovery science that can eventually culminate in new medical therapies. By better understanding the role of XPO1 in RNA export, we can better design therapies that modulate nuclear export or RNA splicing for a wide range of diseases where RNA regulation may play a role.

项目摘要 泰勒实验室的研究重点是真核细胞的核输出，我们通过 扰乱蛋白质的主要输出者XPO1或Exportin-1的正常功能。我们使用分子生物学， 基因组学、蛋白质组学和小鼠模型以确定XPO1介导的核输出机制 XPO1的功能及其在疾病发病机制中的作用我们还研究了XPO1如何与其他蛋白质相互作用 以及核糖核酸(RNA)等分子。在未来五年，该实验室的目标是定义 XPO1在RNA的核出口中的作用。该实验室目前正在进行以下项目。定义 野生型和突变型XPO1对基因表达、剪接和翻译的分子效应。鉴于已知的 XPO1在小核糖核酸和核糖体核糖核酸结合输出中的作用 结合蛋白，我们假设XPO1的变化可能会影响RNA剪接和/或mRNA翻译。 我们已经建立了几个基因工程模型来允许XPO1的内源性表达 E571K或R749Q突变，包括条件性敲击小鼠。我们已经通过各种不同的 XPO1 E571K影响其货物识别的蛋白质组学、生化、结构和分子研究 核输出信号(Nes)并特异性地导致核转录因子κB和转录因子NFAT转录因子的异常输出 蛋白质。我的第一个项目的目标是继续研究XPO1 E571K如何改变核能的程度 输出，并探索RNA输出，以及随后的mRNA剪接和翻译是否受到 RNA结合蛋白的错误定位。我们最近发现了另一个突变，XPO1 R749Q， 通过增加核外蛋白质的出口来影响核出口。我们计划研究哪些蛋白质 这影响和如何影响细胞生长、细胞周期和其他动态平衡过程。我们也在做模特 XPO1的过度表达，并将比较这个过度活跃的突变体与相关的过度表达的情况 蛋白质和RNA的出口。选择性抑制XPO1的药物将使我们能够分离XPO1- 从属出口。此外，我们的初步数据显示，由于SF3B1的异常剪接的细胞 突变在暴露于XPO1抑制剂后发生细胞凋亡。我们还将研究XPO1的影响 基因工程细胞株和SF3B1敲打小鼠对SF3B1表达突变细胞的抑制作用 模特。我们假设XPO1抑制干扰了RNA的输出，并影响了基因表达和mRNA 剪接给出了XPO1通过RNA结合蛋白输出小核RNA的已知作用。这项工作将 为将来提交研究XPO1的生物学及其在 核糖核酸的核出口。这项研究的总体目标与NIGMS的使命是一致的 支持新的基础发现科学，最终可能导致新的医学疗法。变得更好 了解XPO1在RNA输出中的作用，我们可以更好地设计调节核输出或 RNA剪接可用于多种疾病，在这些疾病中，RNA调控可能发挥作用。