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Cell Mar 2023RADAR is a two-protein bacterial defense system that was reported to defend against phage by "editing" messenger RNA. Here, we determine cryo-EM structures of the RADAR...
RADAR is a two-protein bacterial defense system that was reported to defend against phage by "editing" messenger RNA. Here, we determine cryo-EM structures of the RADAR defense complex, revealing RdrA as a heptameric, two-layered AAA+ ATPase and RdrB as a dodecameric, hollow complex with twelve surface-exposed deaminase active sites. RdrA and RdrB join to form a giant assembly up to 10 MDa, with RdrA docked as a funnel over the RdrB active site. Surprisingly, our structures reveal an RdrB active site that targets mononucleotides. We show that RdrB catalyzes ATP-to-ITP conversion in vitro and induces the massive accumulation of inosine mononucleotides during phage infection in vivo, limiting phage replication. Our results define ATP mononucleotide deamination as a determinant of RADAR immunity and reveal supramolecular assembly of a nucleotide-modifying machine as a mechanism of anti-phage defense.
Topics: Bacteriophages; Cryoelectron Microscopy; ATPases Associated with Diverse Cellular Activities; Adenosine Triphosphate; Adenosine Deaminase
PubMed: 36764290
DOI: 10.1016/j.cell.2023.01.012 -
Cell Stem Cell Mar 2023Adenosine deaminase acting on RNA1 (ADAR1) preserves genomic integrity by preventing retroviral integration and retrotransposition during stress responses. However,...
Adenosine deaminase acting on RNA1 (ADAR1) preserves genomic integrity by preventing retroviral integration and retrotransposition during stress responses. However, inflammatory-microenvironment-induced ADAR1p110 to p150 splice isoform switching drives cancer stem cell (CSC) generation and therapeutic resistance in 20 malignancies. Previously, predicting and preventing ADAR1p150-mediated malignant RNA editing represented a significant challenge. Thus, we developed lentiviral ADAR1 and splicing reporters for non-invasive detection of splicing-mediated ADAR1 adenosine-to-inosine (A-to-I) RNA editing activation; a quantitative ADAR1p150 intracellular flow cytometric assay; a selective small-molecule inhibitor of splicing-mediated ADAR1 activation, Rebecsinib, which inhibits leukemia stem cell (LSC) self-renewal and prolongs humanized LSC mouse model survival at doses that spare normal hematopoietic stem and progenitor cells (HSPCs); and pre-IND studies showing favorable Rebecsinib toxicokinetic and pharmacodynamic (TK/PD) properties. Together, these results lay the foundation for developing Rebecsinib as a clinical ADAR1p150 antagonist aimed at obviating malignant microenvironment-driven LSC generation.
Topics: Mice; Animals; Protein Isoforms; Hematopoietic Stem Cells; Adenosine Deaminase
PubMed: 36803553
DOI: 10.1016/j.stem.2023.01.008 -
Nature Jul 2022The RNA-editing enzyme ADAR1 is essential for the suppression of innate immune activation and pathology caused by aberrant recognition of self-RNA, a role it carries...
The RNA-editing enzyme ADAR1 is essential for the suppression of innate immune activation and pathology caused by aberrant recognition of self-RNA, a role it carries out by disrupting the duplex structure of endogenous double-stranded RNA species. A point mutation in the sequence encoding the Z-DNA-binding domain (ZBD) of ADAR1 is associated with severe autoinflammatory disease. ZBP1 is the only other ZBD-containing mammalian protein, and its activation can trigger both cell death and transcriptional responses through the kinases RIPK1 and RIPK3, and the protease caspase 8 (refs. ). Here we show that the pathology caused by alteration of the ZBD of ADAR1 is driven by activation of ZBP1. We found that ablation of ZBP1 fully rescued the overt pathology caused by ADAR1 alteration, without fully reversing the underlying inflammatory program caused by this alteration. Whereas loss of RIPK3 partially phenocopied the protective effects of ZBP1 ablation, combined deletion of caspase 8 and RIPK3, or of caspase 8 and MLKL, unexpectedly exacerbated the pathogenic effects of ADAR1 alteration. These findings indicate that ADAR1 is a negative regulator of sterile ZBP1 activation, and that ZBP1-dependent signalling underlies the autoinflammatory pathology caused by alteration of ADAR1.
Topics: Adenosine Deaminase; Animals; Caspase 8; Cell Death; Gene Deletion; Immune System Diseases; Inflammation; Mammals; Mutation; Protein Kinases; RNA, Double-Stranded; RNA-Binding Proteins; Receptor-Interacting Protein Serine-Threonine Kinases; Signal Transduction
PubMed: 35859177
DOI: 10.1038/s41586-022-04896-7 -
Nature Communications May 2023Targetable drivers governing 5-fluorouracil and cisplatin (5FU + CDDP) resistance remain elusive due to the paucity of physiologically and therapeutically relevant...
Targetable drivers governing 5-fluorouracil and cisplatin (5FU + CDDP) resistance remain elusive due to the paucity of physiologically and therapeutically relevant models. Here, we establish 5FU + CDDP resistant intestinal subtype GC patient-derived organoid lines. JAK/STAT signaling and its downstream, adenosine deaminases acting on RNA 1 (ADAR1), are shown to be concomitantly upregulated in the resistant lines. ADAR1 confers chemoresistance and self-renewal in an RNA editing-dependent manner. WES coupled with RNA-seq identify enrichment of hyper-edited lipid metabolism genes in the resistant lines. Mechanistically, ADAR1-mediated A-to-I editing on 3'UTR of stearoyl-CoA desaturase (SCD1) increases binding of KH domain-containing, RNA-binding, signal transduction-associated 1 (KHDRBS1), thereby augmenting SCD1 mRNA stability. Consequently, SCD1 facilitates lipid droplet formation to alleviate chemotherapy-induced ER stress and enhances self-renewal through increasing β-catenin expression. Pharmacological inhibition of SCD1 abrogates chemoresistance and tumor-initiating cell frequency. Clinically, high proteomic level of ADAR1 and SCD1, or high SCD1 editing/ADAR1 mRNA signature score predicts a worse prognosis. Together, we unveil a potential target to circumvent chemoresistance.
Topics: Humans; Adaptor Proteins, Signal Transducing; Adenosine Deaminase; Cisplatin; DNA-Binding Proteins; Fluorouracil; Proteomics; RNA; RNA Editing; RNA-Binding Proteins; Stearoyl-CoA Desaturase; Stomach Neoplasms; Drug Resistance, Neoplasm
PubMed: 37208334
DOI: 10.1038/s41467-023-38581-8 -
Nature Aug 2022A major challenge in human genetics is to identify the molecular mechanisms of trait-associated and disease-associated variants. To achieve this, quantitative trait...
A major challenge in human genetics is to identify the molecular mechanisms of trait-associated and disease-associated variants. To achieve this, quantitative trait locus (QTL) mapping of genetic variants with intermediate molecular phenotypes such as gene expression and splicing have been widely adopted. However, despite successes, the molecular basis for a considerable fraction of trait-associated and disease-associated variants remains unclear. Here we show that ADAR-mediated adenosine-to-inosine RNA editing, a post-transcriptional event vital for suppressing cellular double-stranded RNA (dsRNA)-mediated innate immune interferon responses, is an important potential mechanism underlying genetic variants associated with common inflammatory diseases. We identified and characterized 30,319 cis-RNA editing QTLs (edQTLs) across 49 human tissues. These edQTLs were significantly enriched in genome-wide association study signals for autoimmune and immune-mediated diseases. Colocalization analysis of edQTLs with disease risk loci further pinpointed key, putatively immunogenic dsRNAs formed by expected inverted repeat Alu elements as well as unexpected, highly over-represented cis-natural antisense transcripts. Furthermore, inflammatory disease risk variants, in aggregate, were associated with reduced editing of nearby dsRNAs and induced interferon responses in inflammatory diseases. This unique directional effect agrees with the established mechanism that lack of RNA editing by ADAR1 leads to the specific activation of the dsRNA sensor MDA5 and subsequent interferon responses and inflammation. Our findings implicate cellular dsRNA editing and sensing as a previously underappreciated mechanism of common inflammatory diseases.
Topics: Adenosine; Adenosine Deaminase; Alu Elements; Autoimmune Diseases; Genetic Predisposition to Disease; Genome-Wide Association Study; Humans; Immune System Diseases; Immunity, Innate; Inflammation; Inosine; Interferon-Induced Helicase, IFIH1; Interferons; Quantitative Trait Loci; RNA Editing; RNA, Double-Stranded; RNA-Binding Proteins
PubMed: 35922514
DOI: 10.1038/s41586-022-05052-x -
Science (New York, N.Y.) Jul 2019Programmable RNA editing enables reversible recoding of RNA information for research and disease treatment. Previously, we developed a programmable adenosine-to-inosine...
Programmable RNA editing enables reversible recoding of RNA information for research and disease treatment. Previously, we developed a programmable adenosine-to-inosine (A-to-I) RNA editing approach by fusing catalytically inactivate RNA-targeting CRISPR-Cas13 (dCas13) with the adenine deaminase domain of ADAR2. Here, we report a cytidine-to-uridine (C-to-U) RNA editor, referred to as RNA Editing for Specific C-to-U Exchange (RESCUE), by directly evolving ADAR2 into a cytidine deaminase. RESCUE doubles the number of mutations targetable by RNA editing and enables modulation of phosphosignaling-relevant residues. We apply RESCUE to drive β-catenin activation and cellular growth. Furthermore, RESCUE retains A-to-I editing activity, enabling multiplexed C-to-U and A-to-I editing through the use of tailored guide RNAs.
Topics: Adenosine; Adenosine Deaminase; Cytidine; Cytosine Deaminase; HEK293 Cells; Humans; Inosine; Protein Domains; Protein Engineering; RNA Editing; RNA-Binding Proteins; Uridine; beta Catenin
PubMed: 31296651
DOI: 10.1126/science.aax7063 -
The New England Journal of Medicine May 2021Severe combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency.
BACKGROUND
Severe combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency.
METHODS
We treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human . Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up.
RESULTS
Overall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade.
CONCLUSIONS
Treatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.).
Topics: Adenosine Deaminase; Adolescent; Agammaglobulinemia; Child; Child, Preschool; Genetic Therapy; Genetic Vectors; Hematopoietic Stem Cell Transplantation; Humans; Infant; Lentivirus; Lymphocyte Count; Progression-Free Survival; Prospective Studies; Severe Combined Immunodeficiency; Transplantation, Autologous
PubMed: 33974366
DOI: 10.1056/NEJMoa2027675 -
Cell Host & Microbe Aug 2021Necroptosis mediated by Z-nucleic-acid-binding protein (ZBP)1 (also called DAI or DLM1) contributes to innate host defense against viruses by triggering cell death to...
Necroptosis mediated by Z-nucleic-acid-binding protein (ZBP)1 (also called DAI or DLM1) contributes to innate host defense against viruses by triggering cell death to eliminate infected cells. During infection, vaccinia virus (VACV) protein E3 prevents death signaling by competing for Z-form RNA through an N-terminal Zα domain. In the absence of this E3 domain, Z-form RNA accumulates during the early phase of VACV infection, triggering ZBP1 to recruit receptor interacting protein kinase (RIPK)3 and execute necroptosis. The C-terminal E3 double-strand RNA-binding domain must be retained to observe accumulation of Z-form RNA and induction of necroptosis. Substitutions of Zα from either ZBP1 or the RNA-editing enzyme double-stranded RNA adenosine deaminase (ADAR)1 yields fully functional E3 capable of suppressing virus-induced necroptosis. Overall, our evidence reveals the importance of Z-form RNA generated during VACV infection as a pathogen-associated molecular pattern (PAMP) unleashing ZBP1/RIPK3/MLKL-dependent necroptosis unless suppressed by viral E3.
Topics: Adenosine Deaminase; Cell Death; Humans; Necroptosis; Protein Kinases; RNA, Double-Stranded; RNA-Binding Proteins; Receptor-Interacting Protein Serine-Threonine Kinases; Vaccinia virus; Viral Proteins
PubMed: 34192517
DOI: 10.1016/j.chom.2021.05.009 -
Molecular Cancer Nov 2020Emerging evidence has shown that circular RNAs (circRNAs) play a crucial regulatory role in the occurrence and development of cancer. Exploring the roles and mechanisms...
BACKGROUND
Emerging evidence has shown that circular RNAs (circRNAs) play a crucial regulatory role in the occurrence and development of cancer. Exploring the roles and mechanisms of circRNAs in tumorigenesis and progression may help to identify new diagnostic markers and therapeutic targets. In the present study, we investigated the role and regulatory mechanism of hsa_circ_0004872 in gastric cancer (GC).
METHODS
qRT-PCR was used to determine the expression of hsa_circ_0004872 in GC tissues and cells. EdU, CCK-8, transwell and scratch wound healing assays were used to assess the role of hsa_circ_0004872 in GC cell proliferation, invasion and migration, respectively. Subcutaneous and tail vein tumor injections in nude mice were used to assess the role of hsa_circ_0004872 in vivo. RIP assay, biotin-coupled probe pull-down assay, FISH and luciferase reporter assay were performed to confirm the relationship between hsa_circ_0004872 and the identified miRNA. ChIP assay, luciferase reporter assay and western blot were used to determine the direct binding of Smad4 to the promoter of the ADAR1 gene.
RESULTS
In this study, we found that hsa_circ_0004872 was dramatically downregulated in GC tissues compared with adjacent noncancerous tissues. The expression level of hsa_circ_0004872 was associated with tumor size and local lymph node metastasis. Enforced expression of hsa_circ_0004872 inhibited the proliferation, invasion and migration of GC cells, whereas knockdown of hsa_circ_0004872 had the opposite effects. Nude mice experiments showed that ectopic expression of hsa_circ_0004872 dramatically inhibited tumor growth and metastasis in vivo. Moreover, we demonstrated that hsa_circ_0004872 acted as a "molecular sponge" for miR-224 to upregulate the expression of the miR-224 downstream targets p21 and Smad4. Importantly, we found that the RNA-editing enzyme ADAR1 inhibited hsa_circ_0004872 expression and further led to the upregulation of miR-224. Smad4, the downstream target of miR-224, could further affect hsa_circ_0004872 levels by directly binding to the promoter region of ADAR1 to inhibit ADAR1 expression.
CONCLUSIONS
Our findings showed that hsa_circ_0004872 acted as a tumor suppressor in GC by forming a negative regulatory loop consisting of hsa_circ_0004872/miR-224/Smad4/ADAR1. Thus, hsa_circ_0004872 may serve as a potential biomarker and therapeutic target for GC.
Topics: Adenosine Deaminase; Animals; Apoptosis; Biomarkers, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Middle Aged; Prognosis; RNA, Circular; RNA-Binding Proteins; Smad4 Protein; Stomach Neoplasms; Survival Rate; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 33172486
DOI: 10.1186/s12943-020-01268-5 -
Redox Biology Nov 2023Astrocytes affect stroke outcomes by acquiring functionally dominant phenotypes. Understanding molecular mechanisms dictating astrocyte functional status after brain...
Astrocytes affect stroke outcomes by acquiring functionally dominant phenotypes. Understanding molecular mechanisms dictating astrocyte functional status after brain ischemia/reperfusion may reveal new therapeutic strategies. Adenosine deaminase acting on RNA (ADAR1), an RNA editing enzyme, is not normally expressed in astrocytes, but highly induced in astrocytes in ischemic stroke lesions. The expression of ADAR1 steeply increased from day 1 to day 7 after middle cerebral artery occlusion (MCAO) for 1 h followed by reperfusion. ADAR1 deficiency markedly ameliorated the volume of the cerebral infarction and neurological deficits as shown by the rotarod and cylinder tests, which was due to the reduction of the numbers of activated astrocytes and microglia. Surprisingly, ADAR1 was mainly expressed in astrocytes while only marginally in microglia. In primary cultured astrocytes, ADAR1 promoted astrocyte proliferation via phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Furthermore, ADAR1 deficiency inhibited brain cell apoptosis in mice with MCAO as well as in activated astrocyte-conditioned medium-induced neurons in vitro. It appeared that ADAR1 induces neuron apoptosis by secretion of IL-1β, IL-6 and TNF-α from astrocytes through the production of reactive oxygen species. These results indicated that ADAR1 is a novel regulator promoting the proliferation of the activated astrocytes following ischemic stroke, which produce various inflammatory cytokines, leading to neuron apoptosis and worsened ischemic stroke outcome.
Topics: Mice; Animals; Astrocytes; Phosphatidylinositol 3-Kinases; Brain Ischemia; Infarction, Middle Cerebral Artery; Neurons; Brain Injuries; Ischemic Stroke; Apoptosis; Reperfusion Injury; Adenosine Deaminase
PubMed: 37801857
DOI: 10.1016/j.redox.2023.102903