Editing of Cellular Self-RNAs by Adenosine Deaminase ADAR1 Suppresses Innate Immune Stress Responses

Abstract

Adenosine deaminases acting on double-stranded RNA (ADARs) catalyze the deamination of adenosine (A) to produce inosine (I) in double-stranded (ds) RNA structures, a process known as A-to-I RNA editing. dsRNA is an important trigger of innate immune responses, including interferon (IFN) production and action. We examined the role of A-to-I RNA editing by two ADARs, ADAR1 and ADAR2, in the sensing of self-RNA in the absence of pathogen infection, leading to activation of IFN-induced, RNA-mediated responses in mouse embryo fibroblasts. IFN treatment of Adar1(-/-) cells lacking both the p110 constitutive and p150 IFN-inducible ADAR1 proteins induced formation of stress granules, whereas neither wild-type (WT) nor Adar2(-/-) cells displayed a comparable stress granule response following IFN treatment. Phosphorylation of protein synthesis initiation factor eIF2α at serine 51 was increased in IFN-treated Adar1(-/-) cells but not in either WT or Adar2(-/-) cells following IFN treatment. Analysis by deep sequencing of mouse exonic loci containing A-to-I-editing sites revealed that the majority of editing in mouse embryo fibroblasts was carried out by ADAR1. IFN treatment increased editing in both WT and Adar2(-/-) cells but not in either Adar1(-/-) or Adar1(-/-) (p150) cells or Stat1(-/-) or Stat2(-/-) cells. Hyper-edited sites found in predicted duplex structures showed strand bias of editing for some RNAs. These results implicate ADAR1 p150 as the major A-to-I editor in mouse embryo fibroblasts, acting as a feedback suppressor of innate immune responses otherwise triggered by self-RNAs possessing regions of double-stranded character.

Keywords: RNA editing; adenosine deaminase acting on RNA (ADAR); innate immunity; interferon; protein kinase RNA-activated (PKR); stress granule.

FIGURE 1.

Stress granule formation is enhanced by IFN treatment of Adar1^−/− cells but not Adar2^−/− or wild-type cells. MEF cells, either wild-type (WT) or Adar null mutant, were left untreated (Unt) or were treated with IFN as indicated and then analyzed for stress granule formation. A, WT, Adar1^−/−, and Adar2^−/− mutant MEF cells analyzed by immunofluorescence microscopy as described under “Experimental Procedures” using antibody to G3BP1 as a marker for SG formation. IFN treatment was with 1000 units/ml of IFNαA/D. B, quantification of SG-positive WT, Adar1^−/−, and Adar2^−/− mutant cells, untreated or IFNαA/D-treated as indicated. Three representative wide field ×40 images were selected, and a minimum of 500 cells was examined for the presence or absence of SG as described under “Experimental Procedures.” Results are expressed as the percentage of cells positive for SG. Results are mean values and standard errors from three independent experiments. Statistical significance was determined by Student's t test; **, p < 0.05; ND, not detected. C, quantification of SG-positive WT, Adar1^−/−, and Adar2^−/− MEF cells, either untreated or IFN-treated as described under B, except treatment was with IFNβ at the indicated concentration.

FIGURE 2.

Stress granule formation is enhanced by IFN treatment of Adar1^−/−p150 cells but not Pkr^−/− or Stat2^−/− cells. MEF cells, either wild-type (WT) or null mutant as indicated, were left untreated (Unt) or were treated with IFN and then analyzed for stress granule formation. A, Adar1^−/−p150, Pkr^−/−, and Stat2^−/− mutant MEF cells were analyzed by immunofluorescence microscopy as described under “Experimental Procedures” using antibody to G3BP1 as a marker for SG formation. IFN treatment was with 1000 units/ml of IFNαA/D. B, quantification of SG-positive Adar1^−/−p150, Pkr^−/−, and Stat2^−/− mutant MEF cells, either untreated or IFNαA/D-treated as indicated, as described for Fig. 1B. Statistical significance was determined by Student's t test; **, p < 0.05; ***, p < 0.005; ND, not detected. C, quantification of SG-positive Adar1^−/−p150, Pkr^−/−, and Stat2^−/− mutant MEF cells, either untreated or IFN-treated as described under Fig. 1B, except treatment was with IFNβ at the indicated concentration.

FIGURE 3.

Phosphorylation of eIF2α in ADAR1-deficient cells following interferon treatment correlates with enhanced stress granule formation. A, relative level of Ser-51 phospho-eIF2α in wild-type (WT) and Adar1^−/− and Adar2^−/− mutant MEF cells, either untreated or treated with IFNβ, was determined by quantification of Western blots using a LI-COR Odyssey infrared imager system. Whole-cell extracts were prepared and analyzed by immunoblot assay with antibodies against eIF2α, phospho_T446 P-eIF2α, and actin. Upper, representative blot. Lower, results shown are mean values and standard errors from three independent experiments. Phospho-eIF2α is expressed relative to total eIF2α protein, normalized to β-actin as the loading control. B, Western immunoblot analysis for PKR expression. Whole-cell extracts were prepared from WT cells or mutant Adar1^−/−, Adar2^−/−, Pkr^−/−, or Adar1^−/−p150 MEF cells, either untreated or IFN-treated, and analyzed by Western blotting using antibodies against PKR and β-actin. Upper, representative blot. Lower, results shown are mean values and standard errors from independent experiments. Total PKR protein amount is relative, normalized to β-actin as the loading control. ND, not detected.

FIGURE 4.

IFN treatment enhances A-to-I editing in MEFs by canonical STAT1/2-dependent signaling, and ADAR1 is responsible for most of the observed editing, both constitutive and inducible. Editing profiles were determined for RNA isolated from untreated and IFNαA/D-treated MEF cells: A, wild-type (WT); B, Adar1^−/−; C, Adar2^−/−; D, Adar1^−/−p150; E, Stat1^−/−; and F, Stat2^−/−. IFN treatment was with 1000 units/ml for 24 h. Microfluidics-based multiplex PCR and deep sequencing were carried out as described under “Experimental Procedures” for 557 mouse exonic loci that contain 11,103 editing sites. The editing levels for sites in IFN-treated cells compared with untreated cells are shown.

FIGURE 5.

Clustered A-to-I editing occurs within regions of predicted secondary structure of cellular transcripts. RNA secondary structure predicted for unedited RNA within 200 nucleotides flanking the PCR amplicon for RPA1 (A) and DNAJC1 (B) transcripts that possess multiple A-to-I-editing sites. The region denoted by the bars corresponds to the enlarged image region and shows edited adenosines that largely cluster on one strand of the predicted duplex region of RPA1 but on both strands of DNAJC1.

FIGURE 6.

Model for the role of ADAR1 as a suppressor of cytoplasmic dsRNA sensing. dsRNA sensors mediate the production of type I interferons and proinflammatory cytokines through activation of IRF3 and IRF7 and NF-κB transcription factors. The sensors of dsRNA include the following: the cytosolic RIG-I-like receptor (RLR) family of proteins (RIG-I, Mda5, and LGP2) that signal via the MAVS (IPS-1 and VISA) mitochondrial adaptor protein and the endosomal membrane-associated Toll-like receptor 3 (TLR3) that signals via the TRIF adaptor. ADARs catalyze the deamination of A in duplex RNA structures producing I that base-pairs as G. ADAR1 p150 is cytoplasmic, whereas ADAR1 p110 and ADAR2 are nuclear enzymes. A-to-I editing leading to I:U mismatch base pairs destabilizes dsRNA structures, both in cellular (self) dsRNAs and viral (non-self) dsRNAs, thereby resulting in reduced levels of functional dsRNA and the impaired activation of dsRNA-dependent sensors. In the absence of ADAR1 p150, dsRNA accumulates and subsequently triggers dsRNA-dependent responses, including activities by RIG-I-like receptors and PKR. Viral infection leads to elevated levels of dsRNA (non-self) compared with levels of cellular (self) dsRNA present in uninfected cells.