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

Our prior analysis of SOX targets in cells identified the human LIMD1 mRNA, which codes for a protein essential for P body formation and integrity, as being highly susceptible to cleavage by SOX (20). The minimum sequence required to directly cut the putative cleavage site in LIMD1 in cells was mapped to a 54 nucleotide segment (LIMD1 54), and we therefore chose this as our model substrate to study SOX targeting in vitro (20) . We first expressed and purified KSHV SOX to greater then 95 percent purity from SF9 insect cells ( fig. S1A ). Using the LIMD1 54 substrate, we plotted the observed rate constant (k obs ) as a function of SOX concentration, yielding a Hill coefficient of n = 1.11 ( Fig. 1A) . Thus, in agreement with previous observations (9, 10) , SOX appears to function predominantly as a monomer. Under conditions of half maximal activity (2 µM; Fig. 1A ), SOX displayed a strong preference for the "hard" divalent metal Mg 2+ and a weaker preference for the "softer" and larger metals Mn 2+ , Co 2+ , and Zn 2+ (Fig. 1B) . This is again consistent with other characterized members of the P/DExK family of enzymes (9, 22) .