Abstract
Inflammation, a central component of the immune response, is produced through a variety of intracellular signalling pathways. A key protein, Retinoic acid-Inducible Gene I (RIG-I), mediates one of these pathways, and is activated by viral RNA. RIG-I signalling is tightly controlled; when regulation goes wrong, autoimmunity can arise. Attachment of a small protein named Ubiquitin is commonly used to confer degradation of protein substrates. Following the resolution of infection, Really Interesting New Gene Finger Protein 125 (RNF125) attaches ubiquitin to RIG-I. RIG-I is then degraded and signalling ends.
This project aims to investigate the interplay between RNF125 and RIG-I. The viral RNA mimic, polyinosinic:polycytidylic acid (poly I:C), was transfected into RNF125-/- and wild-type HEK293 cells to activate RIG-I signalling. RT-qPCR was then used to measure interferon β (IFNB) expression, which is induced by RIG-I signalling. As RIG-I is an interferon stimulated gene, we also investigated RIG-I expression by RT-qPCR and western blotting.
Here, we show that RNF125-/- cells express much more IFNB than wild-type cells when stimulated with poly I:C. Notably, IFNB expression declines in both cell lines, suggesting that signalling terminates even without RNF125. Western blotting of unstimulated cells reveals that RNF125-/- cells have a higher abundance of RIG-I protein compared to wild-type cells (n=3, Student’s t-test, P = 0.0019). Interestingly, mRNA transcript abundance does not differ between cell lines (n = 3, Student’s t-test, P = 0.3226). Poly I:C stimulation increases RIG-I protein and mRNA expression in both cell lines.
Together, these results show that RNF125 controls both basal RIG-I levels and active signalling. Knockout of RNF125 enhances RIG-I signalling and inflammation. This research provides insights into the resolution of immune signalling and how dysregulation of RNF125 and RIG-I contribute to disease.