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.

项目概要 泰勒实验室的研究重点是真核细胞的核输出，我们通过 扰乱蛋白质主要输出蛋白 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 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 在未来五年中，实验室的目标是确定其作用。 RNA 核输出中的 XPO1 目前正在开展以下项目。 已知野生型和突变型 XPO1 对基因表达、剪接和翻译的分子影响。 XPO1 通过 RNA 结合在小核 RNA (snRNA) 和核糖体 RNA (rRNA) 输出中的作用 结合蛋白，我们发现 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基因工程模型来允许 XPO1 的内源表达 E571K或R749Q突变，包括条件敲入小鼠，我们已经通过多种方式证明了。 XPO1 E571K 影响对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) 并特别导致 NFκB 和 NFAT 转录因子的输出改变 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。 输出并探讨 RNA 输出以及随后的 mRNA 剪接和翻译是否受到以下因素的影响 我们最近发现了另一种突变，XPO1 R749Q，它导致 RNA 结合蛋白的错误定位。 通过增加细胞核内蛋白质的输出来影响核输出 我们计划研究哪些蛋白质。 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 的过度表达，并将比较这种过度活跃的突变体与过度表达的关系 选择性抑制 XPO1 的药物将使我们能够分离 XPO1-的影响。 此外，我们的初步数据表明，SF3B1 导致细胞出现异常剪接。 暴露于 XPO1 抑制剂后，突变会发生细胞凋亡。我们还将研究 XPO1 的影响。 使用基因工程细胞系和 Sf3b1 敲入小鼠抑制突变 Sf3b1 表达细胞 我们假设 XPO1 抑制会扰乱 RNA 输出并影响基因表达和 mRNA。 鉴于 XPO1 通过 RNA 结合蛋白输出小核 RNA 的已知作用，这项工作将进行剪接。 作为未来研究 XPO1 生物学及其作用的资助申请的基础数据 RNA 的核输出。这项研究的总体目标与 NIGMS 的使命是一致的。 支持新的基础科学发现，最终可以通过更好的方式实现新的医学疗法。 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 剪接适用于 RNA 调节可能发挥作用的多种疾病。