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

In cells, SOX cleaves its mRNA substrates site-specifically. Mutagenesis of residues in mapped cleavage sites generally abolishes SOX cleavage at that location (20) . To determine if our in vitro assay faithfully recapitulated the site specificity of SOX endonucleolytic targeting observed in cells, we established reaction conditions that enabled trapping of the early cleavage events. By combining Ca 2+ and Mg 2+ in our reaction buffer, we were able to sufficiently slow SOX processing to visualize cleavage products derived from 5' 32 P labeled substrates. Indeed, we observed a predominant 27 nt band, which is the size of the product released upon LIMD1 54 cleavage at the predicted cut site (Fig. 6A, lane 3) . Additional bands also appeared, likely representing subsequent processing events. Importantly, when we incubate SOX with the cut site mutant LIMD1 A-G, there is a complete loss of this 27 nt product, as well as the additionally processed intermediates (Fig. 6A, lane 4) . Production of these cleavage intermediates required SOX, as no decay was observed in the RNA-only controls (Fig. 6A , lanes 1-2).