Mechanistic insights into RNA binding and RNA-regulated RIG-I ubiquitination by TRIM25

Haubrich, Kevin; Augsten, Sandra; Álvarez, Lucía; Huppertz, Ina; Simon, Bernd; Perez, Kathryn; Masiewicz, Pawel; Lethier, Mathilde; Rittinger, Katrin; Gabel, Frank; Hentze, Matthias W.; Cusack, Stephen; Hennig, Janosch

TRIM25 is a ubiquitin E3 ligase active in innate immunity and cell fate decisions. Mounting evidence suggests that TRIM25′s E3 ligase activity is regulated by RNAs. However, while mutations affecting RNA binding have been described, neither the precise RNA binding site has been identified nor which domains are involved. Here, we present biophysical evidence for the presence of RNA binding sites on both TRIM25 PRY/SPRY and coiled-coil domains, and map the binding site on the PRY/SPRY with residue resolution. Cooperative RNA-binding of both domains enhances their otherwise transient interaction in solution and increases the E3 ligase activity of TRIM25. We also show that TRIM25 not only binds RNA in mammalian cells but that interfering with RNA binding has an effect on cellular RIG-I ubiquitination.