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

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/200410 doi: bioRxiv preprint PRY/SPRY domain as the point of contact with the RNA. This discrepancy could arise from the fact that deletion of the CC domain from TRIM25 eliminates its dimerization (Li et al., 2014; Sanchez et al., 2014; Streich et al., 2013) and oligomerization (Koliopoulos et al., 2016 ) ability (Fig. 2c) . While it is not univocally confirmed that TRIM25 needs to dimerize to bind RNA, such a phenomenon is widespread across many RNA-binding proteins (Feracci et al., 2016; Lunde et al., 2007) . Additionally, the experiments presented in Kwon et al. were performed in wild-type mouse Embryonic Stem Cells (mESC), where levels of endogenous TRIM25 are particularly high (Kwon et al., 2013) . This means that any ectopically overexpressed TRIM25 mutant (if it has the CC domain), including TRIM25ΔPRY/SPRY, will have a chance to heterodimerize with the wild-type, endogenous TRIM25. This could mask the requirements for a TRIM25 domain, such as PRY/SPRY, to bind to RNA. Our RNA pull-down data with the extracts from the TRIM25 KO cells clearly demonstrated that T7-TRIM25ΔPRY/SPRY could not bind RNA (Fig. 1g) . Further structural characterization of the TRIM25/RNA complex should reveal novel features of this interaction and shed more light on this novel, putative RNA-binding domain.