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

Importantly, some RNA-binding proteins must dimerize to be able to bind RNA (Feracci et al., 2016; Lunde et al., 2007) . Since TRIM25 is known to dimerize and tetramerize through its CC domain with minimal contributions from the PRY/SPRY (Koliopoulos et al., 2016; Li et al., 2014; Sanchez et al., 2014; Streich et al., 2013) , we decided to generate TRIM25 knockout (KO) cells, which removed any confounding effects of endogenous TRIM25. To obtain TRIM25 KO cells we used CRISPR/Cas9 targeting followed by clonal selection and dot blot analysis. This strategy yielded several HeLa cell clones with complete loss of TRIM25 expression ( Figure S2a ). For further analysis, we selected clone C3 ( Figure S2b ).