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

To test if RNA was necessary for efficient ubiquitination of ZAP, a physiologically important TRIM25 target, we performed in vitro ubiquitination assays of ZAP (ZAPS) with the TRIM25 constructs described earlier (Fig. 6a) . Incubation of T7-ZAP with T7-TRIM25 or T7-TRIM25ΔRBD IPed from HeLa cells with recombinant proteins from the ubiquitin pathway revealed efficient time-dependent ubiquitination of ZAP by TRIM25 but not TRIM25ΔRBD (Fig. 8b) . Notably, to see efficient ubiquitination of ZAP we had to use higher concentrations of recombinant proteins from the ubiquitin pathway than those used for TRIM25 autoubiquitination experiments. As a result, TRIM25 autoubiquitination was much more pronounced in experiments with ZAP ( Fig. 8a) than seen in the case of TRIM25 only assays (Fig. 6b) . Furthermore, it has been recently reported that ZAP enhances TRIM25 autoubiquitination . Next, to show that the observed ZAP ubiquitination was specific and dependent on the activity of TRIM25, we assayed T7-TRIM25ΔRING and T7-TRIM25K117R. TRIM25ΔRING but not T7-TRIM25K117R lost the ability to ubiquitinate T7-ZAP (Fig. 8b) . Intriguingly, we observed autoubiquitination of T7-TRIM25K117R, which All rights reserved. No reuse allowed without permission.