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.
mRNA decay is a widely used mechanism to respond to the need for rapid alteration of gene expression in order to adapt sudden environmental changes such as stress. We found that ADAR1 protects anti-apoptotic gene transcripts from Satufen1/UPF1 mediated mRNA decay and suppresses apoptosis of stressed cells. 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 relevant to the oncogenic transformation process. Therefore, our proposal to expand our previous studies and define the exact function of ADAR1 in the stress response mRNA decay mechanism is highly relevant to cancers.
