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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 and transfected it into TRIM25 KO cells. We also used a T7-TRIM25ΔRING_3'UTR construct that contained the TRIM25 3'UTR. Notably, we had to use more T7-TRIM25ΔRING_3'UTR plasmid to achieve protein expression comparable to that of T7-TRIM25ΔRING (Fig. 5c ). This is most likely connected to microRNA/3'UTR-mediated control of protein expression. As predicted, T7-TRIM25ΔRING does not support efficient autoubiquitination (Fig. 5c ). However, upon EGFP-TRIM25 overexpression, we noticed the appearance of ubiquitinated forms of T7-TRIM25ΔRING ( Fig. 5c-5d ). We could not detect abundant ubiquitination bands in T7-TRIM25ΔRING when EGFP-TRIM25ΔRBD was cooverexpressed ( Fig. 5c-5d ). Even so, the ratio between the ubiquitinated and main bands still showed a positive value, most likely due to weaker expression of T7-TRIM25ΔRING in the presence of EGFP-TRIM25ΔRBD and to background noise. Importantly, when we cooverexpressed the T7-TRIM25ΔRING_3'UTR construct with EGFP-TRIM25, we observed a noticeable increase in the ratio of ubiquitinated vs non-ubiquitinated bands for TRIM25ΔRING, when compared with the T7-TRIM25ΔRING construct without the 3'UTR ( Fig. 5c-5d ). To prove that the observed modification of TRIM25 was specific to K117 we performed similar analyses with T7-TRIM25ΔRING_K117R constructs. We did not observe modification of T7-TRIM25ΔRING_K117R in the absence or presence of EGFP-TRIM25 ( Figure S8c ). Altogether, these results strongly suggest that RNA elements such as the 3'UTR, as well as TRIM25 binding to the RNA, contribute to TRIM25 ubiquitination.