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 these variants in an RRS null (DRRS) animal, using the UAS-Gal4 system. The DRRS line was itself generated using CRISPR Cas9 genome editing, using a dual guide-RNA system. Both RRSWT and RRSSCR rescue the DRRS lethality. Adult animals expressing RRSWT and RRSSCR were compared and contrasted for their response to bacterial infection. Interestingly, RRSSCR animals show an upregulated immune response upon infection, in comparison to RRSWT, as measured by the activation of defence genes using quantitative RNA sequencing. This suggests that SUMOylation of RRS is necessary to restrain aberrant NFkB signalling upon an immune challenge. My research highlights the significance of SUMO conjugation of tRNA synthetases in host defence and uncovers a non-canonical role for SUMOylation of RRS, a member of the MARS complex, in the Drosophila immune response.