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

A key open question related to SOX function is how it can target the majority of mRNAs in cells, yet with significant site specificity. These observations suggest that there must be specific mRNA features that influence targeting. Indeed, PARE-seq analyses of cleavage intermediates in SOX expressing cells revealed that cleavage sites were associated with a degenerate sequence motif (20). Sequences proximal to the cleavage . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 site were predicted to be un-base paired and frequently contained a polyadenosine stretch followed by a purine (20). The importance of these sequence features for SOX targeting was validated for the LIMD1 transcript in cells (20). Because LIMD1 has been established as a particularly robust SOX target in cells (20), we reasoned that it must contain features optimal for SOX processing and therefore would be an ideal substrate to dissect these features biochemically. Indeed, SOX binding to LIMD1 54 was 10-fold better than to the commonly used reporter substrate GFP, and an order of magnitude better than to the K2-31 pre-miRNA, which has not been demonstrated to be processed by SOX in cells. Importantly, these binding differences correlated with the efficiency of SOX cleavage in vitro, arguing that the ability to bind the targeting motif is a key step in target recognition. Through RNA footprinting assays, we were able to show that SOX binds to a bulge structure proximal to the cleavage site containing the polyadenosine stretch previously predicted to be important (20). Mutating either the bulge structure (LIMD1 zipper) or maintaining the bulge but mutating the polyadenosine stretch (LIMD1 3xA-G) resulted in an order of magnitude reduction in binding affinity, correlating with a dramatic decrease in cleavage efficiency. Collectively, these data demonstrate that variability in the efficiency of SOX targeting observed in cells is likely due to differences in RNA sequences that mediate SOX binding.