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 泰勒实验室研究的重点是真核细胞中的核出口，我们通过 扰动蛋白质，XPO1或Exportin-1的主要出口剂的正常功能。我们使用分子生物学， 基因组学，蛋白质组学和小鼠建模，以确定XPO1介导的核输出的机制 XPO1在疾病发病机理中的功能和作用。我们还研究XPO1如何与其他蛋白质相互作用 和分子，例如核糖核酸（RNA）。在接下来的五年中，实验室的目标是确定 XPO1在RNA的核输出中。该实验室目前正在追求以下项目。定义 野生型和突变XPO1对基因表达，剪接和翻译的分子效应。鉴于已知 XPO1在小核RNA（SNRNA）和核糖体RNA（rRNA）中的作用通过RNA的结合 结合蛋白，我们假设XPO1的改变可能会影响RNA剪接和/或mRNA翻译。 我们已经生成了几种普遍的工程模型，以允许XPO1的内源性表达 E571K或R749Q突变，包括有条件的敲蛋白小鼠。我们已经通过各种 XPO1 E571K的蛋白质组学，生化，结构和分子研究影响了对其货物的认识 NFκB和NFAT转录因子的出口变化，核出口信号（NES）和结果明确改变 蛋白质。我的第一个项目的目的是继续研究XPO1 E571K如何改变核的程度 出口并探索RNA导出以及随之而来的mRNA剪接和翻译是否受到影响 RNA结合蛋白的错误定位。我们最近发现了另一个突变XPO1 R749Q， 通过增加蛋白质出口到核us中的出口来影响核出口。我们计划研究哪些蛋白 这会影响细胞生长，细胞周期和其他稳态过程。我们也在建模 XPO1的过表达，将比较这种过度活跃的突变体与关系中的过表达相比 蛋白质和RNA输出。选择性抑制XPO1的药物将使我们能够分离XPO1-的作用 依赖出口。此外，我们的初步数据表明，由于SF3B1引起的异常剪接的细胞 暴露于XPO1抑制剂后突变会凋亡。我们还将研究XPO1的影响 使用一般工程的细胞系和SF3B1敲击蛋白小鼠抑制突变体SF3B1表达细胞 模型。我们假设XPO1抑制作用Perturbs RNA导出并影响基因表达和mRNA 剪接鉴于XPO1通过RNA结合蛋白导出小核RNA的已知作用。这项工作将 作为未来赠款提交的基础数据，调查了XPO1的生物学及其在 RNA的核出口。这项研究的总体目标与Nigms的使命保持一致 支持新的基本发现科学，有时可能在新的医疗疗法中达到顶峰。好的 了解XPO1在RNA导出中的作用，我们可以更好地设计调节核出口或的疗法 RNA剪接可为RNA调节起作用的多种疾病。