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 干扰蛋白质的主要输出者XPO 1或Exportin-1的正常功能。我们用分子生物学， 基因组学、蛋白质组学和小鼠模型，以确定XPO 1介导的核输出机制 XPO 1在疾病发病机制中的作用。我们还研究了XPO 1如何与其他蛋白质相互作用 以及核糖核酸（RNA）等分子。在接下来的五年里，该实验室的目标是确定 XPO 1参与核内RNA的输出。该实验室目前正在开展以下项目。定义 野生型和突变型XPO 1对基因表达、剪接和翻译的分子效应。鉴于已知 XPO 1在通过RNA结合输出小核RNA（snRNA）和核糖体RNA（rRNA）中的作用 结合蛋白，我们假设XPO 1的改变可能会影响RNA剪接和/或mRNA翻译。 我们已经产生了几种基因工程模型，以允许XPO 1的内源性表达。 E571 K或R749 Q突变，包括条件性敲入小鼠。我们已经通过各种各样的 蛋白质组学、生物化学、结构和分子研究表明，XPO 1 E571 K影响其货物的识别。 核输出信号（内斯）并特异性导致NFκB和NFAT转录因子的输出改变 proteins.我的第一个项目的目的是继续研究XPO 1 E571 K如何改变核反应堆的程度。 出口，并探讨RNA出口，以及随后的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种突变，XPO 1 R749 Q， 通过增加蛋白质输出到细胞核外来影响细胞核输出。我们计划研究哪些蛋白质 这会影响以及如何影响细胞生长、细胞周期和其他稳态过程。我们也在做模特 XPO 1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。 蛋白质和RNA的输出。选择性抑制XPO 1的药物将使我们能够分离XPO 1的作用- 依赖出口。此外，我们的初步数据显示，由于SF 3B 1而导致异常剪接的细胞 突变在暴露于XPO 1抑制剂时发生凋亡。我们还将研究XPO 1 使用基因工程细胞系和Sf 3b 1敲入小鼠在突变体Sf 3b 1表达细胞中的抑制 模型我们假设，XPO 1抑制干扰RNA输出，影响基因表达和mRNA 考虑到XPO 1通过RNA结合蛋白输出小核RNA的已知作用，这项工作将 作为未来资助申请的基础数据，研究XPO 1的生物学及其在以下方面的作用： RNA的核输出。这项研究的总体目标与NIGMS的使命一致， 支持新的基础发现科学，最终可以在新的医学疗法中达到顶峰。通过更好 了解XPO 1在RNA输出中的作用，我们可以更好地设计调节核输出的疗法， RNA剪接用于多种疾病，其中RNA调节可能发挥作用。