The p150 Isoform of ADAR1 Blocks Sustained RLR signaling and Apoptosis during Influenza Virus Infection.
ABSTRACT: Signaling through retinoic acid inducible gene I (RIG-I) like receptors (RLRs) is tightly regulated, with activation occurring upon sensing of viral nucleic acids, and suppression mediated by negative regulators. Under homeostatic conditions aberrant activation of melanoma differentiation-associated protein-5 (MDA5) is prevented through editing of endogenous dsRNA by RNA editing enzyme Adenosine Deaminase Acting on RNA (ADAR1). In addition, ADAR1 is postulated to play pro-viral and antiviral roles during viral infections that are dependent or independent of RNA editing activity. Here, we investigated the importance of ADAR1 isoforms in modulating influenza A virus (IAV) replication and revealed the opposing roles for ADAR1 isoforms, with the nuclear p110 isoform restricting versus the cytoplasmic p150 isoform promoting IAV replication. Importantly, we demonstrate that p150 is critical for preventing sustained RIG-I signaling, as p150 deficient cells showed increased IFN-? expression and apoptosis during IAV infection, independent of RNA editing activity. Taken together, the p150 isoform of ADAR1 is important for preventing sustained RIG-I induced IFN-? expression and apoptosis during viral infection.
Project description:Transcripts encoding ADAR1, a double-stranded, RNA-specific adenosine deaminase involved in the adenosine-to-inosine (A-to-I) editing of mammalian RNAs, can be alternatively spliced to produce an interferon-inducible protein isoform (p150) that is up-regulated in both cell culture and in vivo model systems in response to pathogen or interferon stimulation. In contrast to other tissues, p150 is expressed at extremely low levels in the brain and it is unclear what role, if any, this isoform may play in the innate immune response of the central nervous system (CNS) or whether the extent of editing for RNA substrates critical for CNS function is affected by its induction. To investigate the expression of ADAR1 isoforms in response to viral infection and subsequent alterations in A-to-I editing profiles for endogenous ADAR targets, we used a neurotropic strain of reovirus to infect neonatal mice and quantify A-to-I editing in discrete brain regions using a multiplexed, high-throughput sequencing strategy. While intracranial injection of reovirus resulted in a widespread increase in the expression of ADAR1 (p150) in multiple brain regions and peripheral organs, significant changes in site-specific A-to-I conversion were quite limited, suggesting that steady-state levels of p150 expression are not a primary determinant for modulating the extent of editing for numerous ADAR targets in vivo.
Project description:BACKGROUND: RNA-specific adenosine deaminase ADAR1 is ubiquitously expressed in a variety of mammalian cells and tissues. Although its physiological importance in non-nervous tissues has been confirmed by analysis of null mutation phenotypes, few endogenous editing substrates have been identified in numerous peripheral tissues and biological function of ADAR1 has not been fully understood. METHODS: A conditional site-specific, ribozyme-based gene knock-down strategy was utilized to study the function of full-length isoform of ADAR1 (p150 protein) in HeLa cell. Double-stable HeLa cell lines were developed by transfecting HeLa Tet-On cells with a pTRE-derived plasmid that can express a hammerhead ribozyme against mRNA of p150 ADAR1 isoform under induction condition. Semi-quantitative RT-PCR and Western blotting were performed to measure the expression of p150 in selected cell clones. Cell proliferation was evaluated by means of MTT assay and growth curve analysis. Cellular morphological changes were observed under light microscope. Flow Cytometry was used for cell cycle analysis. Growth rate of cell transplants in BALB/c nude mice was also investigated. RESULTS: Both HeLa cell proliferation in vitro and the growth rate of transplanted HeLa cell-derived tumors in nude mice in vivo were significantly inhibited due to reduced expression of ADAR1 p150. Additionally, cell cycle analysis showed that cell progression from G1 phase to S phase was retarded in the ADAR1 p150 suppressed cells. CONCLUSION: Our results suggest that normal expression and functioning of p150 ADAR1 is essential for the maintenance of proper cell growth. The mechanisms underlying ADAR1's action might include both editing of currently unknown double-stranded RNAs and interacting with other cellular dsRNA-related processes.
Project description:The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. Although DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity also may be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, whole-transcriptome sequencing of normal, chronic phase, and serially transplantable blast crisis chronic myeloid leukemia (CML) progenitors revealed increased IFN-? pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the IFN-responsive ADAR1 p150 isoform, and a propensity for increased adenosine-to-inosine RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promotes expression of the myeloid transcription factor PU.1 and induces malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a misspliced form of GSK3? implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of blast crisis CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus that exhibits two alternative life cycles: latency and lytic reactivation. During lytic reactivation, host innate immune responses are activated to restrict viral replication. Here, we report that adenosine deaminase acting on RNA 1 (ADAR1) is required for optimal KSHV lytic reactivation from latency. Knockdown of ADAR1 in KSHV latently infected cells inhibits viral gene transcription and viral replication during KSHV lytic reactivation. ADAR1 deficiency also significantly increases type I interferon production during KSHV reactivation. This increased interferon response is dependent on activation of the RIG-I-like receptor (RLR) pathway. Depletion of ADAR1 together with either RIG-I, MDA5, or MAVS reverses the increased IFN? production and rescues KSHV lytic replication. These data suggest that ADAR1 serves as a proviral factor for KSHV lytic reactivation and facilitates DNA virus reactivation by dampening the RLR pathway-mediated innate immune response.
Project description:Mutations in ADAR, which encodes the ADAR1 RNA-editing enzyme, cause Aicardi-Goutières syndrome (AGS), a severe autoimmune disease associated with an aberrant type I interferon response. How ADAR1 prevents autoimmunity remains incompletely defined. Here, we demonstrate that ADAR1 is a specific and essential negative regulator of the MDA5-MAVS RNA sensing pathway. Moreover, we uncovered a MDA5-MAVS-independent function for ADAR1 in the development of multiple organs. We showed that the p150 isoform of ADAR1 uniquely regulated the MDA5 pathway, whereas both the p150 and p110 isoforms contributed to development. Abrupt deletion of ADAR1 in adult mice revealed that both of these functions were required throughout life. Our findings delineate genetically separable roles for both ADAR1 isoforms in vivo, with implications for the human diseases caused by ADAR mutations.
Project description:Type I IFNs play central roles in innate immunity; however, overproduction of IFN can lead to immunopathology. In this study, we demonstrate that adenosine deaminase acting on RNA 1 (ADAR1), an RNA-editing enzyme induced by IFN, is essential for cells to avoid inappropriate sensing of cytosolic RNA in an inducible knockout cell model-the primary mouse embryo fibroblast derived from ADAR1 lox/lox and Cre-ER mice as well as in HEK293 cells. ADAR1 suppresses viral and cellular RNA detection by retinoic acid-inducible gene I (RIG-I) through its RNA binding rather than its RNA editing activity. dsRNA binds to both ADAR1 and RIG-I, but ADAR1 reduces RIG-I RNA binding. In the absence of ADAR1, cellular RNA stimulates type I IFN production without viral infection or exogenous RNA stimulation. Moreover, we showed in the ADAR1-inducible knockout mice that ADAR1 gene disruption results in high-level IFN production in neuronal tissues-the hallmark of Aicardi-Goutières syndrome, a heritable autoimmune disease recently found to be associated with ADAR1 gene mutations. In summary, this study found that ADAR1 limits cytosolic RNA sensing by RIG-I through its RNA binding activity; therefore, ADAR1 suppresses type I IFN production stimulated by viral and cellular RNAs. These results explain why loss of ADARA1 causes IFN induction and also indicates a mechanism for the involvement of ADAR1 in autoimmune diseases such as Aicardi-Goutières syndrome.
Project description:Measles virus (MV), a member of the family Paramyxoviridae and an exclusively human pathogen, is among the most infectious viruses. A progressive fatal neurodegenerative complication, subacute sclerosing panencephalitis (SSPE), occurs during persistent MV infection of the CNS and is associated with biased hypermutations of the viral genome. The observed hypermutations of A-to-G are consistent with conversions catalyzed by the adenosine deaminase acting on RNA (ADAR1). To evaluate the role of ADAR1 in MV infection, we selectively disrupted expression of the IFN-inducible p150 ADAR1 isoform and found it caused embryonic lethality at embryo day (E) 11-E12. We therefore generated p150-deficient and WT mouse embryo fibroblast (MEF) cells stably expressing the MV receptor signaling lymphocyte activation molecule (SLAM or CD150). The p150(-/-) but not WT MEF cells displayed extensive syncytium formation and cytopathic effect (CPE) following infection with MV, consistent with an anti-MV role of the p150 isoform of ADAR1. MV titers were 3 to 4 log higher in p150(-/-) cells compared with WT cells at 21 h postinfection, and restoration of ADAR1 in p150(-/-) cells prevented MV cytopathology. In contrast to infection with MV, p150 disruption had no effect on vesicular stomatitis virus, reovirus, or lymphocytic choriomeningitis virus replication but protected against CPE resulting from infection with Newcastle disease virus, Sendai virus, canine distemper virus, and influenza A virus. Thus, ADAR1 is a restriction factor in the replication of paramyxoviruses and orthomyxoviruses.
Project description:Long double-stranded RNA may undergo hyper-editing by adenosine deaminases that act on RNA (ADARs), where up to 50% of adenosine residues may be converted to inosine. However, although numerous RNAs may undergo hyper-editing, the role for inosine-containing hyper-edited double-stranded RNA in cells is poorly understood. Nevertheless, editing plays a critical role in mammalian cells, as highlighted by the analysis of ADAR-null mutants. In particular, the long form of ADAR1 (ADAR1(p150)) is essential for viability. Moreover, a number of studies have implicated ADAR1(p150) in various stress pathways. We have previously shown that ADAR1(p150) localized to cytoplasmic stress granules in HeLa cells following either oxidative or interferon-induced stress. Here, we show that the Z-DNA-binding domain (Z?(ADAR1)) exclusively found in ADAR1(p150) is required for its localization to stress granules. Moreover, we show that fusion of Z?(ADAR1) to either green fluorescent protein (GFP) or polypyrimidine binding protein 4 (PTB4) also results in their localization to stress granules. We additionally show that the Z? domain from other Z-DNA-binding proteins (ZBP1, E3L) is likewise sufficient for localization to stress granules. Finally, we show that Z-RNA or Z-DNA binding is important for stress granule localization. We have thus identified a novel role for Z-DNA-binding domains in mammalian cells.
Project description:Adenosine deaminase acting on RNA (ADAR) (also known as ADAR1) promotes A-to-I conversion in double-stranded and highly structured RNAs. ADAR1 has two isoforms transcribed from different promoters: ADAR1p150, which is mainly cytoplasmic and interferon-inducible, and constitutively expressed ADAR1p110 that is primarily localized in the nucleus. Mutations in ADAR1 cause Aicardi – Goutières syndrome (AGS), a severe autoinflammatory disease in humans associated with aberrant IFN production. In mice, deletion of ADAR1 or selective knockout of the p150 isoform alone leads to embryonic lethality driven by overexpression of interferon-stimulated genes. This phenotype can be rescued by concurrent deletion of cytoplasmic dsRNA-sensor MDA5. These findings indicate that the interferon-inducible p150 isoform is indispensable and cannot be rescued by the ADAR1p110 isoform. Nevertheless, editing sites uniquely targeted by ADAR1p150 but also mechanisms of isoform- specificity remain elusive. To identify ADAR1 isoform-specific ‘editome’, we transfected A-to-I editing deficient mouse embryonic fibroblasts (MEFs) with ADAR1p150- or ADAR1p110- or RFP-editing negative control. We subjected the samples to RNA sequencing and detected editing at known-editing sites. Overall design: To identify editing sites preferentially edited by ADAR1p150 or p110, we transfected (electroporated) A-to-I editing deficient MEFs (of the genotype: Adar-/-; Adarb1-/-; Gria2R/R) with p150 or p110 ADAR1-isoform. The experiment was conducted in biological triplicates. Transfection with an RFP-expressing vector was included as editase-negative control (1 sample - processed with the first replica).
Project description:The p150 form of the RNA-specific adenosine deaminase ADAR1 is interferon-inducible and catalyzes A-to-I editing of viral and cellular RNAs. We have characterized mouse genomic clones containing the promoter regions required for Adar1 gene transcription and analyzed interferon induction of the p150 protein using mutant mouse cell lines. Transient transfection analyses using reporter constructs led to the identification of three promoters, one interferon-inducible (P(A)) and two constitutively active (P(B) and P(C)). The TATA-less P(A) promoter, characterized by the presence of a consensus ISRE element and a PKR kinase KCS-like element, directed interferon-inducible reporter expression in rodent and human cells. Interferon induction of p150 was impaired in mouse cells deficient in IFNAR receptor, JAK1 kinase or STAT2 but not STAT1. Whereas Adar1 gene organization involving multiple promoters and alternative exon 1 structures was highly preserved, sequences of the promoters and exon 1 structures were not well conserved between human and mouse.