ADAR1-mediated RNA editing is required for thymic self-tolerance and inhibition of autoimmunity

T cells play a crucial role in the adaptive immune system, and their maturation process is tightly regulated. Adenosine deaminase acting on RNA 1 (ADAR1) is the enzyme responsible for adenosine‐to‐inosine RNA editing in dsRNAs, and loss of ADAR1 activates the innate immune sensing response via melanoma differentiation‐associated protein 5 (MDA5), which interprets unedited dsRNA as non‐self. Although ADAR1 is highly expressed in the thymus, its role in the adaptive immune system, especially in T cells, remains elusive. Here, we demonstrate that T cell‐specific deletion of Adar1 in mice causes abnormal thymic T cell maturation including impaired negative selection and autoimmunity such as spontaneous colitis. This is caused by excessive expression of interferon‐stimulated genes, which reduces T cell receptor (TCR) signal transduction, due to a failure of RNA editing in ADAR1‐deficient thymocytes. Intriguingly, concurrent deletion of MDA5 restores thymocyte maturation and prevents colitis. These findings suggest that prevention of MDA5 sensing of endogenous dsRNA by ADAR1‐mediated RNA editing is required for preventing both innate immune responses and T cell‐mediated autoimmunity

The P2 promoter of rat PC gene contains glucose-responsive element(s) (GRE).

<p>A series of 5′-truncated P2-luciferase reporter constructs were transfected into INS-1 832/13 cells. The transfected cells were maintained in RPMI containing normal (5.5 mM) or high (25 mM) glucose for 24 h. The luciferase activity of each construct was normalized to the β-galactosidase activity and expressed as relative luciferase activity. Relative luciferase values obtained from transfected cells maintaining in high glucose medium were presented as fold change relative to those maintaining in normal concentration of glucose, each of which was arbitrarily set as 1. The statistical analysis was conducted using ANOVA test where **P<0.01.</p

Sp1 regulates glucose-induced PC expression through E-box-like element (E2) in P2 promoter of the PC gene.

<p><b>A</b>, The WT-P2(pGL-P2) or its mutation at E2-luciferase reporter constructs (MuE2) with plasmid overexpressing Sp1, Sp3 or empty vector (pcDNA) were transfected into INS-1 832/13 cells before they were maintained in medium containing low (5.5 mM) or high (25 mM) glucose before their luciferase activities were assayed. Relative luciferase activity of cells co-transfected with pGL-P2 (WT) or mutant promoter construct with empty vector (pcDNA), pSp1 or pSp3 maintained at 5.5 mM or 25 mM were normalized with that of cells co-transfected with pGL-P2 and empty vector maintained at 5.5 mM, which was arbitrarily set as 1. The statistical analysis was conducted using ANOVA test. Ψ (P<0.05) and ΨΨ (P<0.01), compared with cells transfected with pGL-P2 and pcDNA maintained at 5.5 mM glucose. # (P<0.05) and ## (P<0.01), compared with cells transfected with MuE2 maintained at 5.5 mM glucose. **P<0.01 (transactivation of WT promoter by Sp1 or Sp3 under 5.5 mM and 25 mM glucose). <b>B</b>, The Sp1-bound chromatin was prepared from INS-1 832/13 cells grown in low (5.5 mM) or high (25 mM) glucose, fragmented and immunoprecipitated with anti-Sp1 antibody and subjected to real time PCR. The fluorescence signals obtained from quantitation of immunoprecipitated fraction was normalized to the input levels. The input fraction was the sonicated Sp1-bound DNA before immunopreripitating with anti-Sp1 antibody. The statistical analysis was conducted by ANOVA test where *,ΨΨ P<0.01. <b>C</b>, Western blot analysis of nuclear (NC) and cysolic (CYT) extracts of INS-1 832/13 cells maintained under 5.5 or 25 mM glucose with anti-Sp1 antibody. Loading controls of the cytosolic and nuclear proteins were assessed by stripping the blot and re-probed with anti-tubulin and anti-lamin B antibody, respectively. <b>D</b>, A representative of Western blot analysis of nuclear extracts of INS-1 832/13 cells maintained in the presence of 5.5 or 25 m glucose with anti-Sp1, anti-phospho Sp1. Control loading was assessed by stripping the blot and re-probed with anti-actin antibody. E, The intensity of total Sp1, phospho-Sp1 bands was quantitated and normalized with that of actin band and shown as the ratios of total Sp1/actin, phospho-Sp1/actin and total Sp1/phospho-Sp1 from three independent experiments. The statistical analysis was conducted using ANOVA test where *P<0.05 **P<0.01.</p

Multiple E-boxes in the P2 promoter of the PC gene function as GREs.

<p><b>A</b>, A series of 25 or 23-nucleotide internal deletions were generated across the −546 to −399 of P2 promoter (Δ1, Δ2, Δ3, Δ4, Δ5 and Δ6). <b>B</b>, The WT P2 promoter construct containing mutation at E1, E2, E3 and whole ChoRE (MuE1, MuE2, MuE3, MuE4 and MuE2&E4) were generated and transiently transfected into INS-1 832/13 cells. The transfected cells were maintained in RPMI containing normal (5.5 mM) or high (25 mM) glucose for 24 h. The luciferase activity of each construct was normalized to the β-galactosidase activity and expressed as relative luciferase activity. Relative luciferase values obtained from transfected cells maintaining in high glucose medium were presented as fold change relative to those obtained from those maintaining in normal concentration of glucose, each of which was arbitrarily set as 1. The statistical analysis was conducted using ANOVA test where *P<0.05; **P<0.01.</p

USF1 and USF2 bind to the E1 in the P2 promoter of the PC gene.

<p><b>A.</b> Nucleotide sequences of M4, M5 and M5 pmut E2 probes. E-box and GC-box are underline. <b>B,</b> EMSA of M4 probe with INS-1 832/13 nuclear extract. Lane 1, M4 probe alone; lane 2, probe incubated with nuclear extract; lanes 3–4, nuclear extracts pre-incubated with anti-USF1 or anti-USF2 antibody before the probes were added into the reaction, respectively. Lanes 5–6, probe incubated with nuclear extract of INS-1 832/13 overexpressing USF1 or USF2, respectively. Lanes 7 and 8, nuclear extracts of INS-1 832/13 overexpressing USF1 pre-incubated with anti-USF1 antibody or overexpressing USF2 pre-incubated with anti-USF2 antibody before the probe was added into the reaction, respectively. Lane 9, INS-1 832/13 nuclear extract pre-incubated with anti-ChREBP antibody before the probe was added into the reaction. <b>C.</b> EMSA of M5 pmut E2 probe with an INS-1 832/13 nuclear extract. Lane 1, M5 pmut E2 probe alone; lane 2, probe incubated with nuclear extract; lanes 3–5, nuclear extracts pre-incubated with anti-USF1, anti-USF2 or anti-ChREBP antibody before the probes were added into the reaction, respectively. Arrows represent DNA-protein complexes.</p

Sp1 and Sp3 bind E-box-like element (E2) <i>in vitro</i>.

<p><b>A</b>, Nucleotide sequence of M4+M5 and location of various E-boxes [E1, E2 (E-box like), E3 and E4]. <b>B</b>, M4+M5 probe was incubated with nuclear extract of INS-1 832/13 cells and subjected to EMSA. Lanes 1, M4+5 probe alone; lanes 2 and 6, probe incubated with nuclear extract; lanes 3–5, nuclear extracts were pre-incubated with anti-USF1, anti-USF2, anti-ChREBP, respectively. Lanes 7–9, nuclear extracts incubated with anti-Sp1, anti-Sp3 antibodies or both before the probes were added into the reaction, respectively. Lanes 10–11, nuclear extracts were pre-incubated with 50-fold excess mutant or wild type unlabeled oligonucleotide before the probes were added into the reaction, respectively. Lane 12, probe incubated in nuclear extract of INS-1 832/13 cells transfected with an empty vector (Empty) or nuclear extract of INS-1 832/13 cells transfected with plasmid over-expressing Sp1 (o/v Sp1) (lane 13) or Sp3 (o/v Sp5) (lane 15). Lanes 14 and 16, nuclear extracts of INS-1 832/13 cells over-expressing Sp1 or Sp3 were pre-incubated with anti-Sp1 or anti-Sp3 antibody, respectively before probe was added into the reaction. Arrows (C1, C2) represent DNA-protein complexes.</p

High glucose enhances binding of USF2 and ChREBP to E4 in the P2 promoter of the PC gene and suppression of Sp1, USF2 and ChREBP expression blunts glucose-induced expression of endogenous PC expression in INS-1 8322/13.

<p><b>A</b>, <i>Top panel</i>, a schematic representation of the P2 promoter region with E-box4 and primer binding sites for quantitative real time PCR indicated. <i>Bottom panel</i>, the USF1-, USF2- or ChREBP-bound chromatin was prepared from INS-1 832/13 cells grown in low (5.5 mM) or high (25 mM) glucose was prepared, immunoprecipitated with their corresponding antibodies and subjected to real time PCR using the primers indicated above. The fluorescence signals obtained from the immunoprecipitated fractions were normalized to those obtained from the input fraction which was the sonicated transcription factor-bound DNA before immunoprecipitating with the antibodies. The statistical analysis was conducted by ANOVA test where **P<0.01 compared between low and high glucose concentrations. ^ΨΨP<0.001 compared with the fraction that was immunoprecipiated with no antibody at both low or high glucose concentration. <b>B</b>, Western blot analysis of nuclear (NC) and cysolic (CYT) extracts of INS-1 832/13 cells maintained under 5.5 or 25 mM glucose with anti- USF2 antibody. Loading controls of the cytosolic and nuclear proteins were assessed by stripping the blot and re-probed with anti-tubulin and anti-lamin B antibodies, respectively. <b>C</b>, INS-1 832/13 cells were mock- or transfected with siRNAs targeted to Sp1, USF1, USF2 and ChREBP. The transfected cells were cultured in the medium containing low (5.5 mM) or high (25 mM) glucose for the next 24 h before the expression of Sp1, USF1, USF2, ChREBP and PC mRNAs was measured by quantitative real time PCR and their expression levels were normalized with that of 18 s rRNA. The values obtained from scramble and each knocked down cells are expressed relative to that obtained from the mocked transfection, which was arbitrarily set as 100%. The values shown are means ± standard deviations of the three independent experiments (n = 3). The statistical analysis was conducted using ANOVA test where *P<0.05; **P<0.01.</p

RNA editing at a limited number of sites is sufficient to prevent MDA5 activation in the mouse brain.

Adenosine deaminase acting on RNA 1 (ADAR1), an enzyme responsible for adenosine-to-inosine RNA editing, is composed of two isoforms: nuclear p110 and cytoplasmic p150. Deletion of Adar1 or Adar1 p150 genes in mice results in embryonic lethality with overexpression of interferon-stimulating genes (ISGs), caused by the aberrant recognition of unedited endogenous transcripts by melanoma differentiation-associated protein 5 (MDA5). However, among numerous RNA editing sites, how many RNA sites require editing, especially by ADAR1 p150, to avoid MDA5 activation and whether ADAR1 p110 contributes to this function remains elusive. In particular, ADAR1 p110 is abundant in the mouse brain where a subtle amount of ADAR1 p150 is expressed, whereas ADAR1 mutations cause Aicardi-Goutières syndrome, in which the brain is one of the most affected organs accompanied by the elevated expression of ISGs. Therefore, understanding RNA editing-mediated prevention of MDA5 activation in the brain is especially important. Here, we established Adar1 p110-specific knockout mice, in which the upregulated expression of ISGs was not observed. This result suggests that ADAR1 p150-mediated RNA editing is enough to suppress MDA5 activation. Therefore, we further created Adar1 p110/Adar2 double knockout mice to identify ADAR1 p150-mediated editing sites. This analysis demonstrated that although the elevated expression of ISGs was not observed, only less than 2% of editing sites were preserved in the brains of Adar1 p110/Adar2 double knockout mice. Of note, we found that some sites were highly edited, which was comparable to those found in wild-type mice, indicating the presence of ADAR1 p150-specific sites. These data suggest that RNA editing at a very limited sites, which is mediated by a subtle amount of ADAR1 p150, is sufficient to prevents MDA5 activation, at least in the mouse brain