Link between Retroelements, Ro and Interferon Biology in Lupus

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Project Summary A variety of nucleic acid ligands have been implicated in the striking Type I Interferon (IFN-I) response observed in lupus and related diseases but which ligands and receptors drive disease are unclear. Amongst putative ligands are retroelements (RE), one class of which, the SINES, was recently shown to bind to the autoantigen, Ro60. Tying this observation to the surprising finding that Ro60 deficient mice get lupus, we will test an explanation for lupus pathogenesis that links disease susceptibility to Ro function. To test the hypothesis that an excess of SINES relative to Ro induces IFN-I and a lupus like disease, we propose: In Aim 1, we will identify the small non coding RNAs that bind to murine Ro60 and we will define the Ro recognition motifs (RRM) using a highly sensitive and specific immunoprecipitation technique called iCLIP. In addition, we will generate mouse embryonic fibroblasts (MEFS) that are deficient in Ro and in one of the key RNA sensors called RIG-I or MDA5 that stimulate IFN-I signaling. These experiments will identify the RNA ligand, motif(s) and corresponding RNA sensors responsible for cell autonomous IFN-I stimulation. In the second Aim, we will ask how Ro60 and RNA binding elements are regulated, in response to lupus relevant environmental triggers. First, using cell based approaches and the MEFS generated in Aim 1, we will de-repress SINES by inhibiting the methylase that is responsible for suppression of SINE transcription in mice. Next, we will expose cells to UVB radiation that has been shown to result in increased SINE expression. Finally, as prompted by our preliminary data, we will examine whether Estrogen (E2) upregulates SINES or reduces Ro expression. The experiments are expected to validate SINES as the key RNA ligand that provokes cell intrinsic IFN-I stimulation and test whether lupus related environmental factors alter expression of the RNA or protein. Following these experiments, we will examine the same environmental factors in Ro deficient mice. Since Ro deficient mice develop a lupus like autoimmunity for which there is no current explanation, we will explore the hypothesis that, when Ro is limiting or SINES are overexpressed, SINES stimulate IFN-I which in turn leads to a lupus like disease in the appropriate genetic background. We expect that UV light will increase IFN-I expression in the skin and that this finding will be markedly exacerbated in Ro deficient female mice. Anti-Ro antibodies are usually the first autoantibody to develop in human lupus – many years before the onset of clinical disease. They are also associated with congenital heart block and UV mediated skin rashes. Through understanding the roles of SINE elements and Ro in triggering specific IFN pathways in lupus, we should be able to explore the pathobiology with much greater insight in human SLE and devise approaches to targeted prevention and treatment.