Regulation of the drosophila innate immune response by sumo conjugation of amino acyl trna synthetases

Abstract

Post-translational modification of a substrate protein by SUMO (Small Ubiquitin-related modifier) can modify its activity, localization, interaction or function. A large number of SUMO targets in cells have been identified by proteomic studies, but the biological roles for SUMO conjugation for most targets remain elusive. Multi-aminoacyl tRNA Synthetase complex (MARS) is a large cytoplasmic 1.2 MDa signalling hub that acts as a sensor and regulator of the immune response. MARS consists of eight Amino-acyl tRNA Synthetases (AARS) and three non-synthetase adaptors (AIMP1-3). Using quantitative proteomics, we have determined that the members of the MARS complex showed enhanced SUMO conjugation in response to an immune challenge (Handu et. al., 2015). Subsequently, I could demonstrate that eight of its eleven members were SUMO conjugated using in-vitro SUMOylation assays. Immunoprecipitation of the MARS complex, followed by mass spectrometry, suggests that the complex was stabilized in response to both gram-positive and gram-negative infection in adult flies, underscoring a role for MARS in the Drosophila immune response. Glutamyl-Prolyl tRNA Synthetase (EPRS), a member of MARS sub-complex I is SUMO conjugated at its WHEP domain, which is involved in non-canonical roles. In mammals, EPRS dissociates from the MARS complex in response to infection, to form a secondary GAIT complex, that regulates translation. In order to study roles for SUMO conjugation of EPRS, I have used CRISPR Cas9 genome editing technology to generate a SUMO conjugation resistant (SCR) variant (EPRSSCR; EPRSK957R, K1063R, K1083R, K1106R, K1198R). The transgenic lines generated were unstable, precluding the exploration of immune regulation in EPRSSCR flies. Arginyl tRNA Synthetase (RRS), a member of sub-complex II of MARS is also SUMO conjugated. A SCR variant (RRSSCR; RRSK147R,K383R) was uncovered by a combination of in-bacto SUMOylation assay with Lys mutagenesis. Transgenic Drosophila lines of RRSWT and RRSSCR were made by expressing