Innate immune detection of nucleic acids within infected cells is essential for the activation of antiviral immunity. However, these same pathways that protect from infection can also trigger autoimmune disease if they are dysregulated. In the prior period of support, we characterized the mechanisms of autoimmune disease caused by Trex1 deficiency in mice. TREX1 gene mutations in humans cause Aicardi-Goutieres Syndrome, a rare and severe autoimmune disease. We defined the genetic pathway linking Trex1 deficiency to autoimmunity and discovered the underlying mechanisms that link aberrant activation of intracellular nucleic acid sensors to autoimmune disease. Here, we focus on another AGS gene, ADAR, that encodes the ADAR1 double-stranded RNA deaminase. We found that Adar-deficient mice, which are embryonic lethal, are rescued from an aberrant interferon (IFN) response and substantially rescued from lethality by simultaneous disruption of the MAVS pathway of intracellular RNA sensing. Moreover, we uncovered novel developmental phenotypes in Adar/Mavs DKO mice that are independent of the role of ADAR1 in regulating the antiviral response. Based on these findings, we formed our central hypothesis: that ADAR1 serves two essential functions by editing distinct pools of endogenous cellular RNAs: one that prevents MAVS-dependent, IFN-mediated pathology, and one that regulates organ development. Further, we propose that these distinct functions are mediated separately by the two isoforms of ADAR1. To test this hypothesis, we will address the following Specific Aims: (1) Define the genetic pathway linking ADAR1 deficiency to the aberrant IFN response. We will determine which RNA-sensing RIG-I-like receptor drives the MAVS dependent IFN response in Adar-/- mice, determine the contributions of these IFNs to disease, and evaluate the role of ADAR1 in immune and developmental regulation in adult mice. (2) Identify the RNA editing targets of ADAR1 in mice and in human cells. In a close collaboration with colleagues at the Institute for Systems Biology, we will apply a novel computational pipeline for identifying RNA edits with unprecedented depth and accuracy, establishing comprehensive maps of ADAR1 editing sites in mouse embryos, in CRISPR-targeted human cell lines that lack ADAR expression, and in samples from AGS patients with ADAR mutations. (3) Define the contributions of ADAR1 isoforms to editing, immunoregulation, and development. We will reconstitute ADAR-deficient human cells with the p110 isoform of ADAR1, with the p150 isoform, or both, and we will explore isoform- specific editing events. We will then use Adar p150-/- mice to test for differential contributions of each ADAR1 isoform in vivo to immune regulation and development. Our novel tools and computational approaches will provide an integrated picture of ADAR1 function in both mice and human cells, with relevance to the disease mechanisms that underlie AGS.
Aicardi-Goutieres Syndrome (AGS) is a rare and severe human autoimmune disease that is caused by mutations in the ADAR gene that encodes the ADAR1 double-stranded RNA deaminase. Very little is known about the roles of ADAR1 in vivo. We will use a combination of Adar-targeted mice, ADAR-disrupted human cell lines, samples from AGS patients with ADAR mutations, and a novel computational biology approach to establish the RNA editing specificity of ADAR1 and its contributions to development and disease.
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