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-Goutieres 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.Author summary RNA is subject to various post-transcriptional modifications, which add the information to regulate the fate of each RNA. One such modification is RNA editing, in which certain adenosine in double-stranded RNAs is converted to inosine by deamination reaction that is catalyzed by adenosine deaminases acting on RNA (ADARs). ADAR1 and ADAR2 are active editing enzymes in mammals. In addition, ADAR1 is composed of nuclear p110 and cytoplasmic p150 isoforms. However, the difference in the targets and the function of each ADAR is not fully understood. Previous studies demonstrate that innate immunity is activated in Adar1 p150 knockout mice, which is not observed in Adar2 knockout mice. Here, we established Adar1 p110 knockout mice. These mutant mice showed high levels of mortality during the early post-natal stages, whereas we demonstrated that this is caused by RNA editing-independent function of ADAR1 p110. We further generated Adar1 p110/Adar2 double knockout mice, in which innate immunity is not activated, although more than 98% of all the editing sites are absent in the brain of these double KO mice. Collectively, ADAR1 150-mediated RNA editing at a very limited sites is sufficient to avoid activation of innate immunity, at least in the mouse brain.