Stress Response Functions of ADAR1 Regulated by MAP Kinases

MAP 激酶调控 ADAR1 的应激反应功能

• 批准号: 10330572
• 负责人: KAZUKO NISHIKURA
• 金额: $ 37.51万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10330572
• 关键词: 3' Untranslated Regions; ADAR1; Adenosine; Adult; Affinity; Affinity Chromatography; Amino Acids; Antibodies; Apoptosis; Apoptotic; Binding; Biological Process; Cellular Stress; Cessation of life; Code; Complex; Cytoplasm; DNA Damage; DRADA2b protein; Double-Stranded RNA; Ectopic Expression; Elements; Embryo; Embryonic Development; Epitopes; Family member; Gene Expression Alteration; Gene Family; Genes; Inosine; Interferons; Knock-in; Knockout Mice; Knowledge; Life; Luciferases; MAP2K6 gene; MAPK1 gene; Malignant Neoplasms; Mediating; Messenger RNA; MicroRNAs; Mitogen-Activated Protein Kinases; Mus; Mutant Strains Mice; Nuclear; Nuclear Export; Oncogenic; Phenotype; Phosphorylation; Process; Protein Isoforms; Proteins; RNA; RNA Binding; RNA Editing; RNA-specific adenosine deaminase 3; RPS6KA5 gene; Research; Retrotransposon; Ribonucleases; Role; Serine; Short Interspersed Nucleotide Elements; Signal Transduction; Streptavidin; Stress; Structure; System; Threonine; Transcript; Untranslated RNA; adenosine deaminase; aptamer; biological adaptation to stress; cell injury; design; environmental change; experimental study; exportin 5; knock-down; mRNA Decay; mutant; p38 Mitogen Activated Protein Kinase; pre-miRNA; prevent

PROJECT SUMMARY RNA editing that converts adenosine to inosine (A-to-I RNA editing) specifically in double-stranded RNAs (dsRNAs) is catalyzed by adenosine deaminases acting on RNA (ADARs). We pioneered in the field of A-to-I RNA editing by identifying the first ADAR gene family member, ADAR1. Although ADAR1 edits select protein coding sequences, its most common targets are non-coding sequences consisting of inverted repeats of retrotransposon elements such as Alu and SINE. There are two ADAR1 isoforms, one, mainly nuclear localized ADAR1p110, and the other, mostly cytoplasmic ADAR1p150. Furthermore, it seems that there are RNA editing-dependent and -independent functions of ADAR1. We recently discovered a new RNA editing-independent function of ADAR1. Stress-induced phosphorylation of three threonines and two serines of ADAR1 by MKK6-p38-MSK1/2 MAP kinases increased the binding affinity of ADAR1p110 to Exportin-5 (Xpo5) and its nuclear export mediated by the Xpo5/RanGTP system. Once translocated to the cytoplasm, ADAR1p110 promoted survival of stressed cells by protecting anti-apoptotic gene transcripts carrying 3'UTR Alu dsRNA from Staufen1/UPF1-mediated mRNA decay (SMD). In this application, we will explore this newly found stress response function of ADAR1. Using a Luciferase expression construct carrying 3'UTR streptavidin-binding RNA aptamers and Alu-dsRNA structure, we will identify the ribonuclease that degrades the stress response SMD target mRNAs, which are otherwise protected by ADAR1. We will characterize the ribonuclease activity and its role in the stress induced SMD mechanism. We will then identify “cargo” dsRNAs, which are exported to the cytoplasm together with the phosphorylated ADAR1p110 by a sequential affinity chromatography using differentially epitope-tagged Xpo5, RanQ96L, and phosphomimetic ADAR1p110 expression constructs. We will determine the function of the dsRNAs in the stress response mechanism by their ectopic expression and knock down experiments. ADAR1 null embryos die at E11-12 due to widespread apoptosis. Using antibodies specific to the ADAR1 phosphorylated at T808, T811, S814, S823, and S825, we will determine when and where the stress induced ADAR1 phosphorylation occurs during embryonic development. We will then create and conduct phenotypic analysis of new ADAR1 knockin mutant mouse lines, ADAR1-T/S-to-A and ADAR1-T/S-to-D. ADAR1-T/S-to-A mice express solely ADAR1-T/S-to-A non-phosphorylatable mutant proteins, whereas ADAR1-T/S-to-D mice express solely phosphomimetic ADAR1 proteins. By comparing phenotypic differences among these new mutant mouse strains, we will evaluate the importance of the ADAR1 stress response function during embryo development and in adult life. Apoptosis of stressed and DNA-damaged cells is one way to prevent their transformation to cancers, and, thus, the stress response function of ADAR1 is very likely to be relevant to oncogenic transformation process.

项目概要 RNA 编辑可将腺苷转化为肌苷（A-to-I RNA 编辑），特别是在双链 RNA 中 (dsRNA) 由作用于 RNA (ADAR) 的腺苷脱氨酶催化。我们是 A-to-I 领域的先驱 通过识别第一个 ADAR 基因家族成员 ADAR1 进行 RNA 编辑。尽管 ADAR1 编辑选择的蛋白质 编码序列，其最常见的目标是由反向重复组成的非编码序列 反转录转座子元件，例如 Alu 和 SINE。 ADAR1 有两种亚型，一种主要是核亚型 局部 ADAR1p110，另一个主要是细胞质 ADAR1p150。此外，似乎还有 ADAR1 的 RNA 编辑依赖和独立功能。 我们最近发现了 ADAR1 的一种新的独立于 RNA 编辑的功能。压力引起的 MKK6-p38-MSK1/2 MAP 激酶对 ADAR1 的三个苏氨酸和两个丝氨酸的磷酸化增加 ADAR1p110 与 Exportin-5 (Xpo5) 的结合亲和力及其由 Xpo5/RanGTP 介导的核输出 系统。一旦转移到细胞质，ADAR1p110 通过保护细胞来促进应激细胞的存活 抗凋亡基因转录本携带来自 Staufen1/UPF1 介导的 mRNA 衰减 (SMD) 的 3'UTR Alu dsRNA。 在此应用中，我们将探索 ADAR1 新发现的应激反应功能。使用 携带3'UTR链霉亲和素结合RNA适体和Alu-dsRNA结构的荧光素酶表达构建体， 我们将鉴定能够降解应激反应 SMD 目标 mRNA 的核糖核酸酶，否则这些核糖核酸酶 受 ADAR1 保护。我们将表征核糖核酸酶活性及其在应激诱导的 SMD 中的作用 机制。然后我们将识别“货物”dsRNA，它们与 使用差异表位标记的 Xpo5，通过连续亲和层析磷酸化 ADAR1p110， RanQ96L 和拟磷 ADAR1p110 表达构建体。我们将确定其功能 通过异位表达和敲除实验研究双链RNA在应激反应机制中的作用。 ADAR1 由于广泛的细胞凋亡，无效胚胎在 E11-12 时死亡。使用 ADAR1 特异性抗体 T808、T811、S814、S823 和 S825 被磷酸化，我们将确定何时何地诱发应激 ADAR1 磷酸化发生在胚胎发育过程中。然后我们将创建并进行表型 分析新的 ADAR1 敲入突变小鼠系 ADAR1-T/S-to-A 和 ADAR1-T/S-to-D。 ADAR1-T/S-to-A 小鼠仅表达 ADAR1-T/S-to-A 非磷酸化突变蛋白，而 ADAR1-T/S-to-D 小鼠 仅表达拟磷 ADAR1 蛋白。通过比较这些新的表型差异 突变小鼠品系，我们将评估 ADAR1 应激反应功能在胚胎过程中的重要性 发展和成年生活。应激和 DNA 损伤细胞的凋亡是预防其发生的一种方法 ADAR1的应激反应功能很可能与癌症的转化有关 致癌转化过程。