G3BP1 methylation by PRMT5 facilitates its recruitment of TBK1 for IRF3 activation during host response to foreign ribonucleic acids

Abstract

Background and aim Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) has been shown to bind cytosolic nucleic acid sensors including retinoic acid-inducible gene I (RIG-I) and cyclic GMP-AMP synthase (cGAS) in innate immunity. As a scaffolding protein enhancing protein-protein and protein-RNA interactions during cellular stress response, we examine whether G3BP1 could bind additional innate immune signaling molecules and investigate the relevance and mechanisms underlying these interactions. Methods To define G3BP1 function in dsRNA-triggered anti-viral signaling and identify additional interacting molecules, we generated siRNA- and CRISPER-Cas9-mediated G3BP1-knockdown and knockout HEK293T cells, conducted biochemical experiments in human and murine cells including co-immunoprecipitation, domain mutagenesis and mapping, as well as LC-MS/MS analyses to study protein post-translational modifications. We also performed p(I:C)-stimulation assays to measure interferon production, pharmacological inhibition studies to determine enzyme-substrate specificity, as well as confocal immunofluorescence microscopy to examine protein subcellular localization. Results Our study reveals a role for G3BP1 in binding TBK1 and IRF3, two important molecules in innate immune signaling leading to the production of IFN-β. Deficiency in G3BP1 reduces TBK1–IRF3 complex formation and affects IRF3 phosphorylation, nuclear translocation, and the production of IFN-β during p(I:C) stimulation while the phosphorylation of TBK1 was largely preserved. We further show that G3BP1 constitutively associates with IRF3 and recruits TBK1 upon p(I:C) stimulation. These findings suggest that G3BP1 acts as a scaffold to recruit activated TBK1 and facilitates its activation of IRF3. Domain mapping experiments indicated that the C-terminal RGG region of G3BP1 is critical for binding IRF3 and TBK1. LC–MS/MS analysis revealed human G3BP1 to be arginine-methylated at R435 and R460 residues upon p(I:C)-stimulation, and mutagenesis experiments indicated that R460 is required for its efficient binding of TBK1. We further show that the arginine methyltransferase PRMT5 associates with and promotes the symmetric arginine-dimethylation of G3BP1 and pharmacological inhibition of PRMT5 impaired G3BP1–TBK1 interaction. Conclusions Together, our data indicate that G3BP1 could bind PRMT5, TBK1 and IRF3 and reveal G3BP1 as a signaling hub that constitutively binds IRF3 and is arginine-methylated by PRMT5 to recruit TBK1 for IRF3 phosphorylation and activation and the induction of interferon production during host antiviral response.