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 将腺苷转化为肌苷的RNA编辑（A至I RNA编辑），特别是在双链RNA中 双链RNA（dsRNA）由作用于RNA的腺苷脱氨酶（ADAR）催化。我们在A-to-I领域处于领先地位 通过鉴定第一个阿达尔基因家族成员ADAR 1进行RNA编辑。虽然ADAR 1编辑选择蛋白质 编码序列，其最常见的靶标是由以下反向重复序列组成的非编码序列： 逆转录转座子元件如Alu和SINE。有两种ADAR 1亚型，一种主要是核 定位的ADAR 1 p110，和其他，主要是细胞质的ADAR 1 p150。此外，似乎有 ADAR 1的RNA编辑依赖和非依赖功能。 我们最近发现了ADAR 1的一种新的RNA编辑独立功能。应力诱发 MKK 6-p38-MSK 1/2 MAP激酶对ADAR 1的三个苏氨酸和两个丝氨酸的磷酸化作用增强 ADAR 1 p110与Exportin-5（Xpo 5）的结合亲和力及其由Xpo 5/RanGTP介导的核输出 系统一旦转移到细胞质中，ADAR 1 p110通过保护应激细胞，促进应激细胞的存活。 携带来自Staufen 1/UPF 1介导的mRNA衰变（SMD）的3 'UTR Alu dsRNA的抗凋亡基因转录物。 在本申请中，我们将探索ADAR 1的这种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3' UTR-dsRNA结构的荧光素酶表达构建体， 我们将鉴定降解应激反应SMD靶mRNA的核糖核酸酶，这些靶mRNA原本是 受ADAR 1保护。我们将描述核糖核酸酶的活性及其在应力诱导SMD中的作用 机制然后，我们将鉴定“货物”dsRNA，其与mRNA一起输出到细胞质。 通过使用差异表位标记的Xpo 5的连续亲和色谱法， RanQ 96 L和磷酸化模拟ADAR 1 p110表达构建体。我们将确定 通过双链RNA的异位表达和敲除实验探讨其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死亡。使用ADAR 1特异性抗体 在T808、T811、S814、S823和S825处磷酸化，我们将确定应激诱导的时间和地点。 ADAR 1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ADAR 1敲入突变小鼠系ADAR 1-T/S-to-A和ADAR 1-T/S-to-D。ADAR 1-T/S转A 小鼠仅表达ADAR 1-T/S-to-A非磷酸化突变蛋白，而ADAR 1-T/S-to-D小鼠 仅表达拟磷酸化ADAR 1蛋白。通过比较这些新品种之间的表型差异， 突变小鼠品系，我们将评估ADAR 1应激反应功能在胚胎发育过程中的重要性。 发展和成人生活。应激和DNA损伤细胞的凋亡是防止其发生的一种方法。 因此，ADAR 1的应激反应功能很可能与癌症的发生有关。 致癌转化过程