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Stress Response Functions of ADAR1 Regulated by MAP Kinases

MAP 激酶调控 ADAR1 的应激反应功能

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

项目总结