Systematic identification and characterization of immunogenic double-stranded RNAs - PROJECT SUMMARY AND ABSTRACT: Cellular innate immune receptors play a vital role in recognizing pathogenic molecules and initiating an innate immune response to combat infections. However, when these receptors mistakenly sense self molecules, it can lead to the development of autoimmune diseases. One such cytosolic receptor, MDA5, forms filaments on viral double-stranded RNA (dsRNA) to activate the antiviral interferon (IFN) signaling pathway. The question arises: how does MDA5 differentiate between self cellular dsRNAs and non-self viral RNAs? Research con- ducted by our team and others suggests that the ADAR1 protein catalyzes Adenosine-to-Inosine (A-to-I) RNA editing on cellular dsRNAs, marking them as self RNAs and preventing erroneous MDA5 sensing. Mouse mod- els deficient in ADAR1 RNA editing are not viable, but their survival to the full lifespan can be rescued by remov- ing MDA5. In humans, rare autoimmune diseases such as Aicardi-Goutieres Syndrome exhibit loss-of-function mutations in ADAR1 and gain-of-function mutations in MDA5. Our recent work has shown that insufficient editing of dsRNAs is associated with presumed dsRNA-mediated inflammation in various autoimmune and inflammatory diseases, including Inflammatory Bowel Disease, Multiple Sclerosis, Parkinson's Disease, and Coronary Artery Disease. In the well-established ADAR1-dsRNA-MDA5 axis, there is an urgent need to identify the specific cel- lular dsRNAs that require ADAR1 editing to evade MDA5 sensing. We define these dsRNAs as immunogenic dsRNAs, as their lack of editing leads to increased immunogenicity. The primary objective of this research is to develop effective approaches to systematically identify and charac- terize immunogenic dsRNAs involved in inflammatory diseases and to investigate how the editing status and expression level of dsRNAs influence their immunogenicity. Firstly, we will utilize a recently developed genetic approach to identify immunogenic dsRNAs in various human cell types that we determine to be most relevant in inflammatory diseases. Subsequently, we will employ genetic, biochemical, and computational methods to thor- oughly characterize and validate these immunogenic dsRNAs. Secondly, we will expand our work on systemat- ically identifying genetic variants associated with editing levels. This expansion will enable us to pinpoint immu- nogenic dsRNAs that colocalize with GWAS loci associated with common autoimmune and inflammatory dis- eases. Additionally, we will develop a predictive model to assess an individual's burden of immunogenic dsRNAs as a cumulative measure. This model will aid in stratifying patients with inflammatory diseases, where impaired dsRNA editing and heightened dsRNA sensing likely contribute to chronic inflammation. In summary, by identi- fying the most crucial dsRNA substrates of ADAR1 and ligands of MDA5, this work will bridge a significant knowledge gap in the field. Furthermore, it will provide important mechanistic insights and therapeutic potential regarding how ADAR1 RNA editing prevents MDA5-mediated dsRNA sensing and innate immunity in both healthy and inflammatory disease contexts.
