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

Recently, a high-resolution crystal structure was solved of SOX bound to a 31nt fragment of the KSHV pre-microRNA K12-2 (K2-31). In this structure, the only observed contacts between SOX and K2-31 occurred between the four active site residues of SOX . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/320929 doi: bioRxiv preprint (Y373, R248, C247, F179) and the UGAAG motif surrounding the cleavage site of the RNA (21). It was therefore hypothesized that no other residues beyond this unpaired UGAAG motif were involved in transcript recognition (21). However, our data showing a 10-fold difference in binding affinity between two in vivo validated SOX substrates suggested that a more extended interaction surface might distinguish optimal from suboptimal RNA substrates. We therefore used RNA footprinting to map the SOX binding sites on LIMD1 54. Indeed, SOX robustly protected 3 LIMD1 54 adenosines (positions 20-24) from RNase I digestion in a dose dependent manner (Fig. 4) . Notably, this mapped binding site is the same one predicted from in vivo PARE-seq data (20). We also observed a modest protection of base 27 (G) located directly adjacent to the predicted cleavage site of SOX, which represents the region detected in the crystal structure of K2-31 bound to SOX. Collectively, these findings suggest that while SOX may interact with residues directly adjacent to the cut site, a more extensive interaction interface exists for its preferred in vivo targets.