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 further verify our findings and test whether other TRIM proteins with PRY/SPRY domains could have RNA-binding potential, we prepared constructs encoding chimeric proteins, where the TRIM25 RBD was substituted with homologous sequences from several TRIM proteins (TRIM5, TRIM21, TRIM27 and TRIM65) (Fig. 7a) . Moreover, we designed a TRIM25 construct with a scrambled amino acid sequence of the TRIM25 RBD (Fig. 7a) . The chimeric TRIM25 protein constructs expressed in HeLa cells showed various levels of modification ( Fig. 7b) . Importantly, neither T7-TRIM25ΔRBD nor T7-TRIM25RBD_src was ubiquitinated (Fig. 7b) . To see if these results translated to RNA-binding efficiency, we performed RNA pull-down assays with pre-let-7a-1 in HeLa cell extracts. Control assays confirmed that T7-TRIM25 could but T7-TRIM25ΔRBD could not bind RNA (Fig. 7c ). All All rights reserved. No reuse allowed without permission.