The molecular dissection of TRIM25's RNA-binding mechanism provides key insights into its antiviral activity
TRIM25 is an RNA-binding ubiquitin E3 ligase with central but poorly understood roles in the innate immune response to RNA viruses. The link between TRIM25's RNA binding and its role in innate immunity has not been established. Thus, we utilized a multitude of biophysical techniques to identify key RNA-binding residues of TRIM25 and developed an RNA-binding deficient mutant (TRIM25-m9). Using iCLIP2 in virus-infected and uninfected cells, we identified TRIM25's RNA sequence and structure specificity, that it binds specifically to viral RNA, and that the interaction with RNA is critical for its antiviral activity. Overall design: iCLIP of wild-type and RNA binding mutant TRIM25 in either mock or SINV infected (9h, MOI 3) conditions. All SINV infected samples and mock WT samples were performed in triplicate with matching triplicate SMI samples. Mock TRIM25-M9 IP was performed in duplicate.