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

TRIM25 E3 ubiquitin ligase is a new RNA-binding protein with a role in Lin28a-mediated uridylation of pre-let-7 transcripts (Choudhury et al., 2014) . Here, we revealed that the RNAbinding activity of TRIM25 is contained in its PRY/SPRY region. We also presented the first high-throughput analysis of the molecular interactomes of TRIM25. We showed that TRIM25 is a bona fide RNA-binding protein that associates with many proteins involved in RNA metabolism and interacts with numerous coding and non-coding transcripts. This suggests that TRIM25 could play a role in the regulation of RNA metabolism. Our data showed that TRIM25 binding to RNA and to the 3'UTR of TRIM25 mRNA in particular can augment its autoubiquitination. Finally, we demonstrated that RNA is an important constituent of TRIM25's ubiquitin ligase activity towards itself and its physiologically relevant protein target ZAP. Recently, ZAP was shown to suppress HIV-1 replication by binding CG-rich regions in viral RNAs (Takata et al., 2017) . Intriguingly, the first paper that described ubiquitin conjugation to substrates showed that tRNA is required for selected ubiquitination reactions (Ferber and Ciechanover, 1986) . We propose a model whereby the ubiquitin ligase activity of TRIM25 is substantially enhanced by using RNA as a scaffold (Fig. 9a) .