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

Two SOX point mutants, P176S and F179A, located in an unstructured region of the protein that bridges domains I and II have been shown to be selectively required for its endonucleolytic processing of RNA substrates (Fig S2A and S2B ) (8, 21) . Structural data indicate that residue F179 forms a stacking interaction with an adenine base in the RNA, likely stabilizing the protein-RNA interaction, while P176 is hypothesized to contribute to structural rearrangements required for F179 engagement (21). We purified both mutants to evaluate their relative RNA processing and RNA binding activity against the optimal LIMD1 54 substrate. Both mutants displayed purity and elution profiles similar to wild type (WT) SOX (see Fig. S1A ). However, the catalytic efficiency of each mutant was >5-fold less than WT SOX (Fig. 2D) . Furthermore, RNA binding was severely perturbed; the binding kinetics of WT SOX for LIMD1 54 are in the single digit nanomolar range (K d = 7 nM), while P176S and F179A display >2 log defects (K d = 702 nM and 831 nM, respectively) ( Fig 2E and S3A-C) . Thus, the large defect in RNA binding likely explains the decreased efficiency of RNA processing. Notably, while there was a dramatic . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 decrease in the relative affinities of the two mutants for LIMD1 54, there was not a complete loss of binding or RNA processing. This could be a result of secondary nonspecific interactions and/or nonspecific exonucleolytic degradation by SOX from the 5' monophosphorylated end of the probe.