Adenosine Deaminase Acting On RNA 1 Research Articles

Coronavirus S protein alters dsRNA accumulation and stress granule formation through regulation of ADAR1-p150 expression.

The precise role of the highly variable coronavirus S protein in modulating innate immune responses remains unclear. In this study, we demonstrated that the mutant strain of swine coronavirus porcine enteric diarrhea virus induced significantly lower levels of double-stranded RNA (dsRNA) accumulation, inhibited protein kinase R (PKR) activation and suppressed stress granule (SG) formation compared with the classical strain. The 29th amino acid at N-terminus of S was identified as the key functional site for regulation of SG formation, and found that mutant S inhibited PKR phosphorylation and SG formation by upregulating adenosine deaminase acting on RNA 1 (ADAR1)-p150. Notably, the Zα domain of ADAR1-p150 was essential for inhibiting SG formation. Upregulation of ADAR1-p150 also reduced accumulation of dsRNA depending on its RNA editing function. Virus rescue confirmed that the mutant carrying a substitution at amino acid 29 failed to induce ADAR1-p150, leading to dsRNA accumulation, PKR activation and SG formation. Interestingly, the latest severe acute respiratory syndrome coronavirus-2 strains exhibit a novel 25PPA27 deletion at N-terminus of S that was also shown to lead to altered ADAR1-p150 expression and SG inhibition. The transcription factor TCF7L2 was identified as a player in S-mediated transcriptional enhancement of ADAR1-p150. This study is the first to clarify the crucial role of N-terminus of S in immune regulation of coronaviruses.

ADAR1 and ZBP1 in Breast Cancer: Pathological Mechanism and Therapeutics

Breast cancer is a heterogeneous disease in which a variety of genetic and environmental factors are involved. Among them, adenosine deaminase acting on RNA 1 (ADAR1) and Z-DNA binding protein 1 (ZBP1) are the factors that have opposite functions in the process of regulating cell viability as well as inflammatory and immunity. Recently, the two factors have been proved to act as a key player in caspase-8-dependent PANotosis and IFN-γ-dependent immunoregulation in the development of most types of cancers. In this review, we discuss the basic functions of ADAR1 and ZBP1 as well as the interactions between them in regulating innate immune responses in breast cancer therapeutics.

An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation

Adar null mutant mouse embryos die with aberrant double-stranded RNA (dsRNA)-driven interferon induction, and Adar Mavs double mutants, in which interferon induction is prevented, die soon after birth. Protein kinase R (Pkr) is aberrantly activated in Adar Mavs mouse pup intestines before death, intestinal crypt cells die, and intestinal villi are lost. Adar Mavs Eifak2 (Pkr) triple mutant mice rescue all defects and have long-term survival. Adenosine deaminase acting on RNA 1 (ADAR1) and PKR co-immunoprecipitate from cells, suggesting PKR inhibition by direct interaction. AlphaFold studies on an inhibitory PKR dsRNA binding domain (dsRBD)-kinase domain interaction before dsRNA binding and on an inhibitory ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA provide a testable model of the inhibition. Wild-type or editing-inactive human ADAR1 expressed in A549 cells inhibits activation of endogenous PKR. ADAR1 dsRNA binding is required for, but is not sufficient for, PKR inhibition. Mutating the ADAR1 dsRBD3-PKR contact prevents co-immunoprecipitation, ADAR1 inhibition of PKR activity, and co-localization of ADAR1 and PKR in cells.

ADAR1 prevents ZBP1-dependent PANoptosis via A-to-I RNA editing in developmental sevoflurane neurotoxicity

It is well established that sevoflurane exposure leads to widespread neuronal cell death in the developing brain. Adenosine deaminase acting on RNA-1 (ADAR1) dependent adenosine-to-inosine (A-to-I) RNA editing is dynamically regulated throughout brain development. The current investigation is designed to interrogate the contributed role of ADAR1 in developmental sevoflurane neurotoxicity. Herein, we provide evidence to show that developmental sevoflurane priming triggers neuronal pyroptosis, apoptosis and necroptosis (PANoptosis), and elicits the release of inflammatory factors including IL-1β, IL-18, TNF-α and IFN-γ. Additionally, ADAR1-P150, but not ADAR1-P110, depresses cellular PANoptosis and inflammatory response by competing with Z-DNA/RNA binding protein 1 (ZBP1) for binding to Z-RNA in the presence of sevoflurane. Further investigation demonstrates that ADAR1-dependent A-to-I RNA editing mitigates developmental sevoflurane-induced neuronal PANoptosis. To restore RNA editing, we utilize adeno-associated virus (AAV) to deliver engineered circular ADAR-recruiting guide RNAs (cadRNAs) into cells, which is capable of recruiting endogenous adenosine deaminases to promote cellular A-to-I RNA editing. As anticipated, AAV-cadRNAs diminishes sevoflurane-induced cellular Z-RNA production and PANoptosis, which could be abolished by ADAR1-P150 shRNA transfection. Moreover, AAV-cadRNAs delivery ameliorates developmental sevoflurane-induced spatial and emotional cognitive deficits without influence on locomotor activity. Taken together, these results illustrate that ADAR1-P150 exhibits a prominent role in preventing ZBP1-dependent PANoptosis through A-to-I RNA editing in developmental sevoflurane neurotoxicity. Application of engineered cadRNAs to rectify the compromised ADAR1-dependent A-to-I RNA editing provides an inspiring direction for possible clinical preventions and therapeutics.

A novel aptamer-antibody sandwich electrochemical sensor for detecting ADAR1 in complex biological samples

Human adenosine deaminase acting on RNA1 (ADAR1) is an adenosine-to-inosine (A-to-I) RNA-editing enzyme involved in various types of cancer progression. ADAR1 has emerged as a novel prognostic biomarker for cancer. This study describes the application of a newly identified 70-nt DNA aptamer (Apt38483) against ADAR1 to develop a portable and simple electrochemical biosensor platform for the rapid and sensitive detection of ADAR1 in cell lysates. We selected an ADAR1-specific DNA aptamer from a randomized 70-nt single-stranded DNA library using a competitive in vitro selection method. ADAR1 in the cell lysate was sandwiched onto a bare carbon working electrode of an electro-chemically printed chip between the ADAR1 antibody and gold nanoparticles (40 nm) conjugated with Apt38483, followed by electrochemical analysis using differential pulse voltammetry (DPV) for sensor demonstration. A highly sensitive change in current was observed for as little as 0.53 nM ADAR1 in human embryonic kidney cell lysate. Thus, the merging of a novel DNA aptamer probe for ADAR1 with an electrochemical transduction method enabled the development of a simple, low-cost, and rapid method for the direct measurement of ADAR1 in cell lysates and indicated great potential for the development of an ADAR1 analysis platform, which would be useful in cancer prognosis.

Abstract 1485: Hallmarks of APOBEC3 mutagenesis in normal, pre-leukemic, and myeloproliferative neoplasm cell populations

Abstract Changes in gene expression and subsequent editing patterns of APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like) cytosine deaminases have been shown to be present in solid tumor cancer evolution, however, the context specificity and mechanisms by which APOBEC3 enzymes promote cancer initiation and progression require further elucidation, especially in the hematopoietic niche. Building on advanced sequencing data of healthy, pre-leukemic, and myeloproliferative neoplasm patient samples, we seek to uncover normal and malignant patterns of APOBEC3C overexpression in hematopoietic cells. Overexpression of lentiviral APOBEC3C and an editase-mutated APOBEC3C in normal cord blood, healthy bone marrow, and MPN patient hematopoietic stem/progenitor cells (HSPCs) allows us to study the effects of deaminase dysregulation in hematopoietic cells. In addition to APOBEC3, we are exploring the upregulation of adenosine deaminase acting on RNA1 (ADAR1), as we have previously shown that these two innate immune deaminases are synchronously upregulated in the high-risk myelofibrosis (MF) stem cell population compared to normal aged bone marrow. These overexpression studies show novel differential gene expression changes, RNA hyper-editing sites, and DNA mutation signatures induced by APOBEC3 mutagenesis, which can be cross-referenced to gene expression and mutation signatures seen in hematopoietic malignancies and solid tumor cancers. In addition to mutation patterns, the regulation of LINE-1 elements has been shown to be another important function of APOBEC3 enzymes. In these studies, we examine the effects of APOBEC3 overexpression on repetitive element expression in hematopoietic cell populations and have found that there are strong cell type and context patterns across the APOBEC3 family and between cell types within the hematopoietic system. We will use this comprehensive study of APOBEC3 deregulation to uncover predictive biomarkers of malignant transformation. Citation Format: Jane Marie Isquith, Thomas Whisenant, Jessica Pham, Ludmil Alexandrov, Catriona Jamieson. Hallmarks of APOBEC3 mutagenesis in normal, pre-leukemic, and myeloproliferative neoplasm cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1485.

Two Novel and Two Recurrent Variants of the ADAR1 Gene in Three Chinese Families with Dyschromatosis Symmetrica Hereditaria.

Dyschromatosis symmetrica hereditaria (DSH) is a rare autosomal dominant inherited pigmentary dermatosis. The gene responsible for DSH has been identified as adenosine deaminase acting on RNA1 (ADAR1). This study aimed to identify the causative variants in the ADAR1 gene in three Chinese families with DSH. Data and blood samples were collected from three Chinese families with DSH. Whole-exome and Sanger sequencing were performed to detect pathogenic gene mutation in the patients. Bioinformatics tools were used to predict the pathogenicity of the variants. Four heterozygous ADAR1 variants were identified, including two novel missense variants c.2369G>C (Arg790Pro), and 503C>T (Pro168Leu), and two previously reported variants: c.3232C>T(R1078C), and c.1472C>G (p.S491X). The novel c.503C>T variant was predicted as "deleterious" (score =-2.704) by PROVEAN, and "probably damaging" (score = 1) by PolyPhen2. The other novel variant c.2369G>C was also predicted as "deleterious" (score =-4.167) by PROVEAN, "probably damaging" (score = 1) by PolyPhen2, and "disease-causing" (p = 0.999) by Mutation Taster. Two novel ADAR1 variants were found in Chinese patients with DSH. This research has expanded the ADAR1 gene database for DSH, enhancing our comprehension of the underlying mechanisms.

Generation of three induced pluripotent stem cell lines from individuals with Aicardi-Goutières syndrome caused by a c.3019G>A (p.G1007R) autosomal dominant pathogenic variant in ADAR1

Mutations in Adenosine deaminase acting on RNA 1 (ADAR1) gene encoding RNA editing enzyme ADAR1 results in the neuroinflammatory leukodystrophy Aicardi Goutières Syndrome (AGS). AGS is an early onset leukoencephalopathy with an exacerbated interferon response leading to neurological regression with intellectual disability, spasticity, and motor deficits. We have generated three induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) of individuals with ADAR1G1007R mutation. The generated iPSCs were investigated to confirm a normal karyotype, pluripotency, and trilineage differentiation potential. The reprogrammed iPSCs will allow us to model AGS, dissect the cellular mechanisms and testing different treatment targets.

ADAR1 regulates macrophage polarization and is protective against liver ischemia and reperfusion injury

Liver ischemia and reperfusion injury (LIRI) is a major risk for the poor prognosis of patients receiving liver transplantation. The molecular mechanism involved in LIRI is complex and related to various cellular components. We previously reported that adenosine deaminase acting on RNA 1 (ADAR1) alleviated the allogeneic skin graft rejection by regulating macrophage polarization. However, the regulatory effects of ADAR1 on liver macrophages after LIRI remain largely unknown. In this study, we mainly adopted a mouse model of LIRI and cellular experiments with hypoxia and reoxygenation (HR) treatment to explore the regulatory roles of ADAR1 on liver macrophages under LIRI conditions. We found that IRI caused decreased ADAR1 in liver tissues and remarkable changes of liver macrophage polarization and profiles. ADAR1 supplementation alleviated the pathological injury caused by IRI and accelerated the activation of M2 macrophages in the liver of IRI mice. Increased hypoxia duration reduced ADAR1 expression levels in murine RAW264.7 macrophages at the transcriptional level. Further overexpression of ADAR1 significantly increased the expressions of anti-inflammatory cytokines and promoted M2 polarization of macrophages under HR exposure. ADAR1 knockdown exhibited opposite effects on macrophage polarization. Hence, ADAR1 promotes the M2 polarization of liver macrophages that may further alleviate LIRI. The protective effects of ADAR1 against LIRI provide a novel insight into the prevention and treatment of LIRI.

Abstract A170: Discovery of small molecule inhibitors of ADAR1

Abstract Background: Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA editing enzyme that catalyzes the deamination of adenosine to inosine in double-stranded (ds) RNA. Cellular dsRNAs are edited by ADAR1 to prevent their recognition by cytoplasmic dsRNA sensors such as MDA5 and PKR. Loss of ADAR1 has been shown to induce the activation of these sensors in cancer cells with high intrinsic type 1 interferon signaling, leading to cell death. Accent has developed a Type I Interferon Stimulated Gene (TISG) score through analysis of publicly available datasets that is predictive of sensitivity to ADAR1 loss. As 15-30% of primary tumors in TCGA display elevated type I interferon signaling, ADAR1 has become an attractive oncology target. Here we describe the development of an in vitro and cellular assay suite that was used to progress chemical matter identified from a high-throughput screen (HTS) into single-digit nanomolar inhibitors of cellular ADAR1. Materials and Methods: A HAP-1 cell model of ADAR1 loss was used in rescue experiments to validate a small molecule inhibition approach towards the targeting of ADAR1 activity. A high-throughput screening-compatible biochemical assay was developed and applied to screen diversity libraries, leading to the identification of a chemical series of ADAR1 inhibitors. The mechanism of inhibition of the series was characterized by substrate-competition and orthogonal substrate binding assays. Iterative optimization of the series resulted in cell-permeable compounds of sufficient potency to be tested in a suite of ADAR1 cellular assays. Results: ADAR1 loss or catalytic site-mutant cells were more sensitive to exogenous Interferon-β than parental cells, leading to decreased growth and viability of HAP-1 cells. We demonstrate knock-out of ADAR1 or loss of catalytic activity results in activation of PKR upon interferon-β treatment in the HAP-1 model, unlike parental cells. Development of a label-free biochemical assay of sufficient sensitivity to be run under steady-state conditions, showing linear reaction progress curves over multiple rounds of ADAR1 turnover, enabled high-throughput screening. A hit series was identified that showed RNA substrate non-competitive behavior in orthogonal RNA binding assays. To further assist understanding of series mechanism, a crystallization system was developed that produced a novel human ADAR1 structure to 1.45Å resolution. A suite of in vitro and cellular assays were used to further optimize and identify small molecules with robust inhibition of cellular ADAR1, as read out by cellular RNA editing assays, as well as activation of downstream dsRNA sensor pathways: secretion of interferon B and phosphorylation of PKR. Consistent with our target hypothesis, treatment of the TISG-high squamous cell carcinoma OE21 cell line with potent ADAR1 inhibitors results in cell growth inhibition. Conclusions: Accent developed a suite of assays that led to the identification of small molecule inhibitors targeting ADAR1. Citation Format: Shane M Buker, Stephen J Blakemore, P. Ann Boriack-Sjodin, Cindy Collins, Robert A Copeland, Kenneth W Duncan, Anna Ericsson, Alexandra Garadino, April Greene-Colozzi, Nathalie Leger, Gordon Lockbaum, Anugraha Raman, Scott Ribich, Allen Sickmier, Brian S Sparling, Jie Wu, Serena Silver. Discovery of small molecule inhibitors of ADAR1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A170.

Abstract A104: ADAR1-associated metabolic vulnerabilities in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC), lacking expression of estrogen, progesterone, and HER2 receptors, is aggressive and lacks targeted treatment options. Therefore, the mission to identify actionable targets remains the top priority. We recently demonstrated that an RNA editing enzyme, adenosine deaminase acting on RNA 1 (ADAR1), plays important roles in TNBC’s ability to initiate and maintain tumorigenesis. We posit that ADAR1 functions as a homeostatic factor protecting TNBC from stresses when otherwise kept in check, contributing to tumorigenesis. By both targeted and unbiased approaches, we identified two metabolic vulnerabilities, 1) iron-dependent cell death pathway, ferroptosis, and 2) serine addiction, associated with ADAR1 in TNBC. By performing single-reaction monitoring-based liquid chromatography coupled to mass spectrometry (LC-MS) to measure intracellular lipid contents, we showed that ADAR1 loss in TNBC cells increased the abundance of polyunsaturated fatty acid phospholipids (PUFA-PL), of which peroxidation is the primary driver of ferroptosis. Transcriptomic analysis and phenotypic drug screen using a ferroptosis-focused library were performed to identify underlying mechanisms and drug candidates synergizing with ADAR1 loss through ferroptosis, respectively. Meanwhile, an unbiased screen using a FDA-approved compound library revealed that ADAR1 loss also sensitizes TNBC cell line MDA-MB231 to inhibition of the serine biosynthesis pathway. We showed that ADAR1 knockdown leads to altered serine transport, and a metabolic flux analysis using labeled (U-13C) glucose will be performed to determine if ADAR1 loss affects de novo serine biosynthesis and one-carbon metabolism. By testing the hypothesis that ADAR1 regulates the metabolic fitness of TNBC by desensitizing ferroptosis and serine addiction, we aim to leverage these metabolic vulnerabilities to inform basic, pre-clinical, and clinical studies to develop novel therapeutic strategies for TNBC. Citation Format: Che-Pei Kung, Leah P Shriver, Nick Terzich, Ma Xenia G Ilagen, Mike Prinsen, Sua Ryu, Raleigh D Kladney, Gary J Patti, Leonard B Maggi, Jason D Weber. ADAR1-associated metabolic vulnerabilities in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A104.

The anti-cancer drug candidate CBL0137 induced necroptosis via forming left-handed Z-DNA and its binding protein ZBP1 in liver cells

CBL0137, a promising small molecular anti-cancer drug candidate, has been found to effectively induce apoptosis via activating p53 and suppressing nuclear factor-kappa B (NF-κB). However, it is still not clear whether CBL0137 can induce necroptosis in liver cancer; and if so, what is the underlying molecular mechanism. Here we found that CBL0137 could significantly induce left-handed double helix structure Z-DNA formation in HepG2 cells as shown by Z-DNA specific antibody assay, which was further confirmed by observing the expression of Z-DNA binding protein 1 (ZBP1) and adenosine deaminase acting on RNA 1 (ADAR1). Interestingly, we found that caspase inhibition significantly promoted CBL0137-induced necroptosis, which was further supported with the increase of the late apoptosis and necrosis assessed by the flow cytometry. Furthermore, we found that CBL0137 can also induce the expression of the three necroptosis-related proteins: receptor interacting serine/threonine kinase 1 (RIPK1), receptor interacting serine/threonine kinase 3 (RIPK3), and mixed lineage kinase domain-like (MLKL). Taken together, it was assumed that CBL0137-indued necroptosis in liver cells was due to induction of Z-DNA and ZBP1, which activated RIPK1/RIPK3/MLKL pathway. This represents the first report on the induction of the Z-DNA-mediated necroptosis by CBL0137 in the liver cancer cells, which should provide new perspectives for CBL0137 treatment of liver cancer.

Anti-Inflammatory Properties of Eugenol in Lipopolysaccharide-Induced Macrophages and Its Role in Preventing β-Cell Dedifferentiation and Loss Induced by High Glucose-High Lipid Conditions.

Inflammation is a natural immune response to injury, infection, or tissue damage. It plays a crucial role in maintaining overall health and promoting healing. However, when inflammation becomes chronic and uncontrolled, it can contribute to the development of various inflammatory conditions, including type 2 diabetes. In type 2 diabetes, pancreatic β-cells have to overwork and the continuous impact of a high glucose, high lipid (HG-HL) diet contributes to their loss and dedifferentiation. This study aimed to investigate the anti-inflammatory effects of eugenol and its impact on the loss and dedifferentiation of β-cells. THP-1 macrophages were pretreated with eugenol for one hour and then exposed to lipopolysaccharide (LPS) for three hours to induce inflammation. Additionally, the second phase of NLRP3 inflammasome activation was induced by incubating the LPS-stimulated cells with adenosine triphosphate (ATP) for 30 min. The results showed that eugenol reduced the expression of proinflammatory genes, such as IL-1β, IL-6 and cyclooxygenase-2 (COX-2), potentially by inhibiting the activation of transcription factors NF-κB and TYK2. Eugenol also demonstrated inhibitory effects on the levels of NLRP3 mRNA and protein and Pannexin-1 (PANX-1) activation, eventually impacting the assembly of the NLRP3 inflammasome and the production of mature IL-1β. Additionally, eugenol reduced the elevated levels of adenosine deaminase acting on RNA 1 (ADAR1) transcript, suggesting its role in post-transcriptional mechanisms that regulate inflammatory responses. Furthermore, eugenol effectively decreased the loss of β-cells in response to HG-HL, likely by mitigating apoptosis. It also showed promise in suppressing HG-HL-induced β-cell dedifferentiation by restoring β-cell-specific biomarkers. Further research on eugenol and its mechanisms of action could lead to the development of therapeutic interventions for inflammatory disorders and the preservation of β-cell function in the context of type 2 diabetes.

Abstract 17142: Deficiency of Smooth Muscle ADAR1 Exacerbates Vascular Remodeling and Pulmonary Hypertension

Background: Adenosine deaminase acting on RNA 1 (ADAR1) catalyzes the conversion of adenosine (A) to inosine (I) in double-stranded RNA, which is critical to prevent auto-inflammatory responses mediated by activation of the type I interferon (IFN) signaling. Here, we define the role of ADAR1-dependent RNA editing in IFNβ activation and pulmonary artery smooth muscle cell (PASMC) remodeling in pulmonary arterial hypertension (PAH), a devastating disease leading to right heart failure and premature death. Methods: Total RNAs from IPAH PASMCs (N=3) and healthy control (N=3) were deep sequenced by RNA-seq and determined the fraction of reads with a ‘G’ nucleotide editing sites using RADAR database. A transgenic line ADAR1 SMC-KO was generated by knocking down ADAR1 selectively in αSMA+ cells, followed by 3 weeks of hypoxia. PKR inhibitor Imoxin was used to treat ADAR1 SMC-KO . Results: SMC layers of human IPAH tissues expressed reduced levels of ADAR1. Moreover, IPAH PASMCs displayed decreased mRNA and protein levels of ADAR1, along with reduced editing sites compared to healthy PASMCs (Fig.1A). The knockdown of ADAR1 in PASMCs resulted in the activation of the MDA5-PKR-IFNβ signaling pathway. Compared with controls in vivo , hypoxic ADAR1 SMC-KO mice developed severe PH, as evidenced by excessive vascular remodeling in distal arterioles (Fig.1B) and increased vascular leakage resulting in elevated right ventricular systolic pressure (37 vs 23 mmHg) and right ventricular hypertrophy by Fulton Index (46 vs. control 20%) (Fig.1C). The immunofluorescence staining showed higher expression of IFNβ and increased recruitment of inflammatory immune cells. Imoxin treatment prevented PH & RVH. Conclusions: The deficiency of ADAR1 leads to the activation of IFNβ thus contributing to the pathological processes observed in PAH. Inhibiting PKR activity could attenuate the aberrant IFNβ activation and potentially serve as a new therapeutic strategy to treat PAH.

ADAR1 suppression causes interferon signaling and transposable element transcript accumulation in human astrocytes.

Neuroinflammation is a central mechanism of brain aging and Alzheimer's disease (AD), but the exact causes of age- and AD-related neuroinflammation are incompletely understood. One potential modulator of neuroinflammation is the enzyme adenosine deaminase acting on RNA 1 (ADAR1), which regulates the accumulation of endogenous double-stranded RNA (dsRNA), a pro-inflammatory/innate immune activator. However, the role of ADAR1 and its transcriptomic targets in astrocytes, key mediators of neuroinflammation, have not been comprehensively investigated. Here, we knock down ADAR1 in primary human astrocytes via siRNA transfection and use transcriptomics (RNA-seq) to show that this results in: (1) increased expression of type I interferon and pro-inflammatory signaling pathways and (2) an accumulation of transposable element (TE) transcripts with the potential to form dsRNA. We also show that our findings may be clinically relevant, as ADAR1 gene expression declines with brain aging and AD in humans, and this is associated with a similar increase in TE transcripts. Together, our results suggest an important role for ADAR1 in preventing pro-inflammatory activation of astrocytes in response to endogenous dsRNA with aging and AD.

Generation of a new Adar1p150-/- mouse demonstrates isoform-specific roles in embryonic development and adult homeostasis.

The RNA editing enzyme Adenosine deaminase acting on RNA 1 (ADAR1) is an essential regulator of innate immune activation by both cellular and viral dsRNA. Adenosine-to-Inosine (A-to-I) editing by ADAR1 modifies the sequence and structure of endogenous dsRNA and masks it from the cytoplasmic dsRNA sensor melanoma differentiation-associated protein 5 (MDA5), preventing innate immune activation. Loss of function mutations in ADAR are associated with rare autoinflammatory disorders including Aicardi-Goutieres Syndrome (AGS), defined by a constitutive systemic upregulation of type I interferon (IFN). The murine Adar gene encodes two protein isoforms with distinct functions: ADAR1p110 is constitutively expressed and localizes to the nucleus, whereas ADAR1p150 is primarily cytoplasmic and is inducible by IFN. Recent studies have demonstrated the critical requirement for ADAR1p150 to suppress innate immune activation by self dsRNAs, however, detailed in vivo characterization of the role of ADAR1p150 during development and in adult mice is lacking. We developed a new ADAR1p150-specific knockout mouse mutant based on a single nucleotide deletion that resulted in the loss of the ADARp150 protein without affecting ADAR1p110 expression. The Adar1p150-/- died embryonically at E11.5-E12.5 due to cell death in the fetal liver accompanied by an activated IFN response. Somatic loss of ADAR1p150 in adults was lethal and caused rapid hematopoietic failure, demonstrating an ongoing requirement for ADAR1p150 in vivo. The generation and characterization of this mouse model demonstrates the essential role of ADAR1p150 in vivo and provides a new tool for dissecting the functional differences between ADAR1 isoforms and their physiological contributions.

ADAR1 polymorphisms contribute to increased susceptibility in pediatric acute lymphoblastic leukemia.

Adenosine deaminase acting on RNA1 (ADAR1), catalyzing post-transcriptional adenosine-to-inosine RNA editing, promotes cancer progression and therapeutic resistance. However, very little is known about the association of ADAR1 variants with acute lymphoblastic leukemia (ALL). Here we first explored the potential association of three polymorphisms (rs9616, rs2229857, and rs1127313) of ADAR1 with susceptibility in Chinese children ALL, then functionally characterized ADAR1 in ALL. Our results demonstrated that rs9616 T and rs2229857 T were associated with increased expression of ADAR1 mRNA and higher risk to ALL. Of note, a stronger risk effect of rs2229857 T genotypes was found among relapse children. Furthermore, ADAR1 knockdown specifically inhibited proliferation and promoted apoptosis in ALL cells. These findings give insights into a mechanism by which the risk variant at rs9616 and rs2229857 modulate ADAR1 expression and confers a predisposition and relapse risk to ALL, and representing a potential novel biomarker for pediatric ALL.

Increased RNA editing sites revealed as potential novel biomarkers for diagnosis in primary Sjögren's syndrome

BackgroundTranscriptome-wide aberrant RNA editing has been shown to contribute to autoimmune diseases, but its extent and significance in primary Sjögren's syndrome (pSS) are currently poorly understood. MethodsWe systematically characterized the global pattern and clinical relevance of RNA editing in pSS by performing large-scale RNA sequencing of minor salivary gland tissues obtained from 439 pSS patients and 130 non-pSS or healthy controls. FindingsCompared with controls, pSS patients displayed increased global RNA-editing levels, which were significantly correlated and clinically relevant to various immune features in pSS. The elevated editing levels were likely explained by significantly increased expression of adenosine deaminase acting on RNA 1 (ADAR1) p150 in pSS, which was associated with disease features. In addition, genome-wide differential RNA editing (DRE) analysis between pSS and non-pSS showed that most (249/284) DRE sites were hyper-edited in pSS, especially the top 10 DRE sites dominated by hyper-edited sites and assigned to nine unique genes involved in the inflammatory response or immune system. Interestingly, among all DRE sites, six RNA editing sites were only detected in pSS and resided in three unique genes (NLRC5, IKZF3 and JAK3). Furthermore, these six specific DRE sites with significant clinical relevance in pSS showed a strong capacity to distinguish between pSS and non-pSS, reflecting powerful diagnostic efficacy and accuracy. ConclusionThese findings reveal the potential role of RNA editing in contributing to the risk of pSS and further highlight the important prognostic value and diagnostic potential of RNA editing in pSS.

RNA editing of AZIN1 coding sites is catalyzed by ADAR1 p150 after splicing

Adenosine-to-inosine RNA editing is catalyzed by nuclear adenosine deaminase acting on RNA 1 (ADAR1) p110 and ADAR2, and cytoplasmic ADAR1 p150 in mammals, all of which recognize dsRNAs as targets. RNA editing occurs in some coding regions, which alters protein functions by exchanging amino acid sequences, and is therefore physiologically significant. In general, such coding sites are edited by ADAR1 p110 and ADAR2 before splicing, given that the corresponding exon forms a dsRNA structure with an adjacent intron. We previously found that RNA editing at two coding sites of antizyme inhibitor 1 (AZIN1) is sustained in Adar1 p110/Aadr2 double KO mice. However, the molecular mechanisms underlying RNA editing of AZIN1 remain unknown. Here, we showed that Azin1 editing levels were increased upon type I interferon treatment, which activated Adar1 p150 transcription, in mouse Raw 264.7cells. Azin1 RNA editing was observed in mature mRNA but not precursor mRNA. Furthermore, we revealed that the two coding sites were editable only by ADAR1 p150 in both mouse Raw 264.7 and human embryonic kidney 293T cells. This unique editing was achieved by forming a dsRNA structure with a downstream exon after splicing, and the intervening intron suppressed RNA editing. Therefore, deletion of a nuclear export signal from ADAR1 p150, shifting its localization to the nucleus, decreased Azin1 editing levels. Finally, we demonstrated that Azin1 RNA editing was completely absent in Adar1 p150 KO mice. Thus, these findings indicate that RNA editing of AZIN1 coding sites is exceptionally catalyzed by ADAR1 p150 after splicing.

An essential role of adenosine deaminase acting on RNA 1 in coeliac disease mucosa.

Type I interferons (IFNs) are highly expressed in the gut mucosa of celiac disease (CD) gut mucosa and stimulates immune response prompted by gluten ingestion, but the processes that maintain the production of these inflammatory molecules are not well understood. Adenosine deaminase acting on RNA 1 (ADAR1), an RNA-editing enzyme, plays a crucial role in inhibiting self or viral RNAs from activating auto-immune mediated responses, most notably within the type-I IFN production pathway. The aim of this study was to assess whether ADAR1 could contribute to the induction and/or progression of gut inflammation in patients with celiac disease. ADAR1 expression was assessed by Real time PCR and Western blotting in duodenal biopsy taken from inactive and active celiac disease (CD) patients and normal controls (CTR). To analyze the role of ADAR1 in inflamed CD mucosa, lamina propria mononuclear cells (LPMC) were isolated from inactive CD and ADAR1 was silenced in with a specific antisense oligonucleotide (AS) and then incubated with a synthetic analogue of viral dsRNA (poly I:C). IFN-inducing pathways (IRF3, IRF7) in these cells were evaluated with Western blotting and inflammatory cytokines were evaluated with flow cytometry. Lastly, the role of ADAR1 was investigated in a mouse model of poly I:C-driven small intestine atrophy. Reduced ADAR1 expression was seen in duodenal biopsies compared to inactive CD and normal controls. Ex vivo organ cultures of duodenal mucosal biopsies, taken from inactive CD patients, stimulated with a peptic-tryptic digest of gliadin displayed a decreased expression of ADAR1. ADAR1 silencing in LPMC stimulated with a synthetic analogue of viral dsRNA strongly boosted the activation of IRF3 and IRF7 and the production of type-I IFN, TNF-α and IFN-γ. Administration of ADAR1 antisense but not sense oligonucleotide to mice with poly I:C-induced intestinal atrophy, significantly increased gut damage and inflammatory cytokines production. These data show that ADAR1 is an important regulator of intestinal immune homeostasis and demonstrate that defective ADAR1 expression could provide to amplifying pathogenic responses in CD intestinal mucosa.