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: 10093094
• 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 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10093094
• 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.

项目摘要 RNA编辑将腺苷转换为插入（A-to-I RNA编辑），专门在双链RNA中 （DSRNA）由作用于RNA（ADAR）的腺苷死亡蛋白催化。我们在A到I的领域开创了 RNA编辑通过识别第一个ADAR基因家族成员ADAR1。尽管ADAR1编辑选择蛋白 编码序列，其最常见的目标是非编码序列 逆转录座子元素，例如Alu和Sune。有两个ADAR1同工型，一个主要是核 局部ADAR1P110，另一个主要是细胞质ADAR1P150。此外，似乎有 RNA编辑依赖性和非依赖性功能。 我们最近发现了ADAR1的新型RNA编辑函数。压力引起的 MKK6-P38-MSK1/2 MAP激酶的三种三辛氨酸和两种ADAR1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。一旦易位到细胞质，ADAR1P110通过保护促进应力细胞的存活 来自Staufen1/UPF1介导的mRNA衰变（SMD）的抗凋亡基因转录本。 在此应用程序中，我们将探索ADAR1的新发现的压力响应功能。使用 荧光素酶表达构建构建具有3'UTR链霉亲和素结合的RNA适体和alu-dsRNA结构， 我们将确定降解压力响应SMD目标mRNA的核糖核酸酶，否则 受adar1保护。我们将表征核糖核酸酶活性及其在应力诱导的SMD中的作用 机制。然后，我们将确定“货物” DSRNA，这些DSRNA与细胞质一起出口 使用不同表位标签的XPO5，通过顺序亲和色谱法磷酸化的ADAR1P110， RANQ96L和磷酸化ADAR1P110表达构建体。我们将确定 DSRNA通过其生态表达和敲低实验在应力反应机制中。 Adar1 由于宽度细胞凋亡，无效的胚胎死于E11-12。使用特定于ADAR1的抗体 在T808，T811，S814，S823和S825处磷酸化，我们将确定应力诱导的何时何地 ADAR1磷酸化发生在胚胎发育过程中。然后，我们将创建和进行表型 分析新的ADAR1敲击突变小鼠系，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不可磷酸磷酸突变突变蛋白，而ADAR1-T/S-TO-D小鼠 表达仅磷酸化ADAR1蛋白。通过比较这些新的表型差异 突变小鼠菌株，我们将评估胚胎中ADAR1应力响应函数的重要性 发展和成人生活。压力和DNA受损细胞的凋亡是防止其 转化为癌症，因此，ADAR1的压力响应功能很可能与 致癌过程。