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 find TRIM25-interacting proteins, we performed co-IPs of T7-tagged TRIM25 in HeLa cells coupled with quantitative SILAC mass spectrometry. In brief, wild-type T7-tagged TRIM25 was expressed in HeLa cells, and the co-IP was performed using protein A agarose beads coupled with the anti-T7 antibody (Fig. 3a) . The control cells (without T7-TRIM25) were grown in "heavy" medium, and the cells expressing T7-tagged TRIM25 were grown in detected in the co-IP with T7-TRIM25 but not T7-TRIM25ΔRBD, although the RNase treatment resulted in only a decreased interaction (Fig. 3e) . Importantly, many proteins that were enriched in the T7-TRIM25ΔRBD and RNase-treated T7-TRIM25 assays were associated with ribosomal and translation functions ( Fig. 3b-3c ). They represent RNAindependent interactions with TRIM25 (Table S1) . Surprisingly, the T7-TRIM25ΔRBD and RNase-treated T7-TRIM25 co-IPs detected several proteins that were not seen in the wildtype T7-TRIM25 co-IP. This could be due to a synthetic effect of the mutation, lack of binding to RNA or unspecific binding events. Altogether, these results reaffirm that the PRY/SPRY domain is important for RNA-binding and show that TRIM25 interacts with many RNA-binding proteins, using RNA as a scaffold.