The immune system protects the body against infection by viruses, bacteria, and other pathogens. Cells have a range of signalling pathways that respond to different stimuli. The Retinoic acid-Inducible Gene I (RIG-I) pathway is activated when viral dsRNA binds to the RIG-I intracellular viral sensor protein. Signalling ultimately leads to cytokine and interferon release, which results in clearance of the virus. This pathway is tightly regulated; dysregulation is associated with autoimmunity. Ubiquitylation is important for activating and terminating RIG-I signalling. Notably, addition of Lys63-linked chains is important for activation of signalling, while RING Finger Protein 125 (RNF125) tags RIG-I with Lys48-linked ubiquitin chains that trigger proteasomal degradation, thus terminating signalling.

Despite being reported as a negative regulator of RIG-I signalling, the regulation of RNF125 itself is poorly characterised. Mutations in RNF125 are associated with Tenorio syndrome, a rare disorder characterised by various features, including intellectual disability, developmental delay, and autoimmunity. Variants associated with the disease are shown to have altered function, specifically impeding degradation of RIG-I. How this relates to the clinical phenotype at the molecular level remains to be understood.

This project aimed to investigate how RNF125, and therefore RIG-I, is regulated. Here, we show that RNF125 modulates both active RIG-I signalling as well as basal RIG-I levels. This function requires the RING domain of RNF125, however, the ubiquitin interacting motif appears less essential. TurboID proximity labelling was used to identify potential interactors that control RNF125 function. Finally, a novel RNF125 variant associated with Tenorio syndrome was characterised – while its activity and stability are not impaired, its ability to bind Lys63-linked ubiquitin chains is reduced.