Abstract
Cells have evolved a range of immune signalling pathways to sense and respond to different pathogens. Retinoic acid-Inducible Gene I (RIG-I) signalling is activated in response to viral dsRNA in the cytosol. The RIG-I protein binds to the dsRNA, triggering immune signalling that ultimately leads to cytokine and interferon release, and clearance of the virus. Once infection is overcome, signalling is terminated. RIG-I signalling is tightly regulated; dysregulation is associated with disease.
Attachment of ubiquitin to RIG-I is required for activation and termination of RIG-I signalling. Ubiquitin can be repeatedly attached onto a substrate to form chains with var- ious functions. Addition of Lys63-linked ubiquitin chains to RIG-I by several E3 ligases is needed to activate signalling. Conversely, attachment of Lys48-linked chains to RIG-I by Really Interesting New Gene (RING) Finger Protein 125 (RNF125) triggers proteasomal degradation and terminates signalling. Regulation of RNF125 itself is poorly understood.
This project aimed to investigate how RNF125, and therefore RIG-I, is regulated. Findings from this study suggested that RNF125 modulates both termination of RIG-I signalling and basal RIG-I protein abundance. RIG-I signalling in RNF125-/- HEK293 cells was considerably enhanced and termination was delayed. Basal RIG-I protein levels were also significantly higher when RNF125 was knocked out. Additionally, expression of the RNF125 gene was upregulated by RIG-I signalling, highlighting a potential negative feedback loop. TurboID proximity labelling was used to identify potential protein interactors that control RNF125 function. However, validation of potential interactors by co-immunoprecipitation and AlphaFold modelling had limited success.
The stability of RNF125 is also regulated by autoubiquitylation. Disruption of key interface residues revealed that the RING, but not the ubiquitin interacting motif (UIM), was essential for ubiquitylation of RIG-I and RNF125. Additionally, autoubiquitylation of RNF125 was found to occur in cis.
Mutations in RNF125 are associated with Tenorio syndrome, a rare neurodevelopmental disorder characterised by intellectual disability, developmental delay, and au- toimmunity. RNF125 variants associated with the disease often impede RIG-I degradation. We recently identified a novel RNF125 variant associated with Tenorio syndrome, (NM 017831.4): c.670G>C p.Glu224Gln, the first variant identified in the UIM. While its activity and stability were not impaired, the variant’s ability to bind Lys63-linked ubiquitin chains was reduced. These results suggested that chain-binding by the UIM is required for the normal function of RNF125 in cells.
Altogether, this thesis demonstrates the key role RNF125 plays in controlling RIG-I abundance and highlights the need to further understand how it is regulated.