RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination

Nila Roy Choudhury; Gregory Heikel; Maryia Trubitsyna; Peter Kubik; Jakub Stanislaw Nowak; Shaun Webb; Sander Granneman; Christos Spanos; Juri Rappsilber; Alfredo Castello; Gracjan Michlewski

KO cells when compared with the wild type cells. Western blot analysis of the wild type and KO cells validated this observation (Fig. 3f) . To see if TRIM25-dependent proteasome degradation of ZAP contributes to its lower levels in the wild type cells we treated the cells with proteasome inhibitor MG132. Upon MG132 treatment we noticed increase of ZAP levels only in the wild type cells but not in the TRIM25 KO cells ( Figure S2c ). The signal corresponding to ubiquitin was elevated in both wild type and TRIM25 KO cells ( Figure S2d ). Importantly, overexpression of T7-TRIM25 but not T7-TRIM25ΔRBD or catalytically inactive T7-TRIM25ΔRING decreased the levels of ZAP in TRIM25 KO cells ( Figure S2e ).