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

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.