The broad aim of this project is to identify possible triggers of systemic lupus erythematosus (SLE) and evaluate if extracellular RNA (exRNA) and ribonucleoproteins (RNPs), which accumulate under stress conditions in cytoplasmic granules, can stimulate innate immune responses and result in the formation of autoantibodies. Genetic mutations in factors facilitating RNP granule formation or inhibiting their resolution may further influence their immunostimulatory properties, including size, structure, or local concentration in tissues. Posttranslational modification of nuclear RBPs and RNA-processing factors including phosphorylation, ubiquitination, arginine dimethylation, and protease cleavage, as well as the associated RNAs and their specific sequences and structures accompanying various maturation stages, may be critical to distinguish normal from pathologic and immunogenic RNPs.
The specific aims are: (1) Characterize the exRNA composition in SLE patients with distinct classes of antibodies. Examine if exRNA composition of cultured immortalized B cells from SLE patients mirrors variations detected in serum exRNA composition. Assay the effect of cellular stress on the immunostimulatory properties of immortalized B cell lysates or tissue culture supernatants obtained under normal and stress conditions, and identify relevant RNPs by fractionation experiments. (2) Examine the influence of extracellular and intracellular RNases on exRNA abundance and composition, in existing mouse models, and investigate if any of these profiles resembles the exRNA composition of SLE patients, arguing for a role of RNases in the pathogenesis of SLE. (3) Develop human cell culture models for the biochemical isolation and characterization of stress-related RNA granules. Test various drugs to reproducibly induce specific stress conditions (e.g. oxidative stress, heat shock or translational arrest) and use fluorescence-based assays to monitor dynamic effects on RNA granule formation. Establish assays for interferon and cytokine activation upon application of the aforementioned stress conditions using cultured cells as well as primary cells.
Considering the recent discovery of extracellular RNA as trigger of innate immunity, the determination of the presence of intra- and extracellular RNAs and RNPs in SLE patients may provide clues with respect to the yet unknown trigger of the disease. We will also establish novel RNAseq and cell-based assays to facilitate the discovery of immunomodulatory RNAs and their associated proteins.
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|Ren, Aiming; Patel, Dinshaw J (2014) c-di-AMP binds the ydaO riboswitch in two pseudo-symmetry-related pockets. Nat Chem Biol 10:780-6|