Site specific target binding controls RNA cleavage efficiency by the Kaposi’s sarcoma-associated herpesvirus endonuclease SOX

Aaron S. Mendez; Carolin Vogt; Jens Bohne; Britt A. Glaunsinger

Endonuclease-directed mRNA degradation plays key roles in the lifecycle of gammaherpesviruses, yet the fundamental principles governing target specificity by SOX are not well understood. Here, through the development of the first biochemical system to faithfully recapitulate the internal cleavage specificity observed for SOX in cells, we revealed how both RNA sequence and structure contribute to targeting. These findings resolve a central feature of the current model of SOX activity ( Figure 7 ). Previous observations established that sequences flanking the cut site were required to direct cleavage by SOX (16, 20) . However, it was unresolved whether they played a strictly structural role in presenting an exposed loop for cleavage, served as a platform for SOX binding, or created a binding site for one or more cellular factors that then indirectly recruited SOX to its targets. Through a combination of mutational analyses, RNA structure probing, and RNA footprinting assays, we showed that efficient SOX targeting requires both an exposed loop structure and upstream sequences that serve as a SOX binding platform. This combination of sequence and structural features within the targeting motif helps explain why some mRNAs are efficiently cleaved by SOX, whereas others are weaker substrates.