Stress Response Functions of ADAR1 Regulated by MAP Kinases

项目总结 专门在双链RNA中将腺苷转化为肌苷的RNA编辑(A-to-I RNA编辑) (DsRNAs)是由作用于RNA的腺苷脱氨酶(ADAR)催化的。我们在A-to-I领域开创了先河 通过鉴定第一个ADAR基因家族成员ADAR1进行RNA编辑。尽管ADAR1编辑了精选蛋白质 编码序列，其最常见的靶标是非编码序列，由 反转录转座子元件，如Alu和Sine。有两种ADAR1亚型，一种主要是核的 定位于ADAR1p110，另一个主要为胞质ADAR1p150。此外，似乎还有一些 ADAR1的RNA编辑依赖和独立功能。 我们最近发现了ADAR1的一个新的不依赖RNA编辑的功能。应激诱导 MKK6-p38-MSK1/2对ADAR1三个苏氨酸和两个丝氨酸的磷酸化增强 ADAR1p110与Exportin-5(Xpo5)的结合亲和力及其由Xpo5/RanGTP介导的核输出 系统。一旦被转移到细胞质，ADAR1p110通过保护 从Staufen1/UPF1介导的mRNA衰退(SMD)中携带3‘UTRAlu dsRNA的抗凋亡基因转录本。 在本应用中，我们将探索ADAR1的这一新发现的应激反应函数。使用 携带3‘非编码链亲和素结合RNA适配子和Alu-dsRNA结构荧光素酶表达载体， 我们将确定降解应激反应SMD靶mRNAs的核糖核酸酶，否则 受ADAR1保护。我们将表征核糖核酸酶的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”dsRNAs，它们与 通过使用差异表位标记的XPO5的顺序亲和层析来磷酸化ADAR1p110， RanQ96L和拟磷ADAR1p110表达载体。我们将确定 DsRNAs在应激反应机制中的异位表达和击倒实验。ADAR1 由于广泛的细胞凋亡，零胚胎在E11-12死亡。使用针对ADAR1的抗体 T808、T811、S814、S823和S825的磷酸化，我们将确定应激诱导的时间和地点 ADAR1的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 新的敲门突变小鼠品系ADAR1-T/S-to-A和Adar-T/S-to-D的分析ADAR1-T/S至A 小鼠只表达ADAR1-T/S-to-A非磷酸化突变蛋白，而ADAR1-T/S-to-D小鼠仅表达非磷酸化突变蛋白 仅表达模拟磷酸化的ADAR1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ADAR1应激反应功能在胚胎中的重要性 在成人生活中也是如此。应激和DNA损伤细胞的凋亡是防止其 转化为癌症，因此，ADAR1的应激反应函数很可能与 致癌转化过程。

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.