Enrichment of Zα domains at cytoplasmic stress granules is due to their innate ability to bind nucleic acids

Zα domains are a subfamily of winged Helix-Turn-Helix (wHTH) domains found exclusively in proteins involved in the nucleic acids sensory pathway of vertebrate innate immune system and host evasion by viral pathogens. Interestingly, they are the only known protein domains that recognise the left-handed helical conformation of both dsDNA and dsRNA, known as Z-DNA and Z-RNA. Previously, it has been demonstrated that ADAR1 and ZBP1, two proteins possessing the Zα domains, localize to cytosolic stress granules. It was further speculated that such localization is principally mediated by Zα domains. To characterize and better understand such distinct and specific localization, we characterised the in vivo interactions and localization pattern for the amino terminal region of human DAI harbouring two Zα domains (ZαβDAI). Using immunoprecipitation and mass spectrometry, we identified several interacting partners that were components of the complex formed by Zα domains and RNAs. Differential interacting partners to wild-type Zα, relative to mutant proteins, demonstrated that most of the physiologically relevant interactions are mediated by the nucleic acid binding ability of the Zαβ. Further, we also show enrichment of selected complex components in cytoplasmic stress granules under conditions of stress. This ability is mostly lost in the mutants of ZαβDAI (ZαβDAI 4×mut) that lack nucleic-acid binding ability. Thus, we posit that the mechanism for the translocation of Zα domain-containing proteins to stress granules is mainly mediated by the nucleic acid binding ability of their Zα domains. Finally, we demonstrate that FUS and PSF/p54nrb, two RNA binding proteins with established roles in stress granules, interact with Zα, which provides strong evidence for a role of these proteins in the innate immune system. ### Competing Interest Statement The authors have declared no competing interest. * ADAR1 : Adenosine deaminases acting on RNA SG : Stress granules NONO : Non-POU domain-containing octamer-binding protein SFPQ : splicing factor proline and glutamine rich G3BP1 : Ras GTPase-activating protein-binding protein 1 DDX5 : DEAD-Box Helicase 5 DDX1 : DEAD-Box Helicase 1 RBM14 : RNA Binding motif Protein 14 LC-MS : Liquid chromatography mass spectrometry DAI : DNA-dependent activator of IFN-regulatory factor