Project Leader: Dinshaw Patel. The broad aim of this project is the biochemical and structural characterization of the natural function of a class of autoantigens that are released upon stress and may trigger strong innate immune responses that ultimately result in autoantibody production. Extracellular RNA is a potent activator of the innate immune system sensed by binding to endosomal and intracellular pattern recognition receptors present in most cells including phagocytic cells. The most well known sources of immunostimulatory extracellular RNA are viruses. However, RNA sensing receptors by themselves cannot discriminate stringently against self-nucleic acids. Thus, events of cellular stress that lead to the uptake of host cell RNA into neighboring cells, which then activates pattern recognition receptors, may represent a plausible mechanism by which autoimmunity is initiated. Autoimmune disorders are characterized by the presence of high titers of antinuclear antibodies (ANA), and may be the pathologic manifestations of inappropriate activation of sensing mechanisms by stress-induced RNA-containing granules. We propose to: (1) Identify the RNA binding targets of RBP autoantigens including SSB/La, TROVE2/SSA2/Ro60 and TRIM21/SSA1/Ro52 under normal and stress conditions using PAR-CLIP. Develop new biochemical assays that recapitulate the formation of RNA granules or their resolution using recombinant proteins. (2) Study the role of pol III RNA transcript binding factors during stress and the specific structure and function of SSB/La, TROVE2/SSA2/RO60 and TRIM21/SSA1/Ro52 with their in vivo RNA targets. Investigate how stress conditions influence the processing patterns of tRNAs, e.g. polyuridylation. (3) Determine the mechanism and structure of pol III RNA transcript processing involving the tRNA-specific endoribonuclease TSN-TSNAX/C3P0 and pattern recognition receptor RIG-1 and MDA5 complexes with their in vivo RNA targets, and investigate their perturbation under stress conditions
Despite the long-established involvement of autoantibodies against RNA-binding proteins (RBPs) in SLE, a systematic effort to define the molecular link between RBPs, their targets, and autoimmune disorders has been lacking. In this project, we have outlined a research program that directly characterizes the biochemical and structural properties of specific RNA-protein complexes (RNPs) that have documented and/or compelling roles in autoimmunity.
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