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-line probing was performed as described previously (1) . Briefly, pBSSK(-) plasmids containing the indicated sequences (see Table S2 ) were linearized by digestion with XhoI and ScaI for GFP or BlpI and SacI (NEB) for LIMD1, gel purified, phenol/chloroform extracted, and ethanol precipitated. The fragments were then used as templates for in vitro transcription with the HiScribe T7 High Yield RNA synthesis Kit (NEB) and afterwards subjected to Turbo DNase (Ambion by Life Technologies) treatment. RNA was revolved by 8% Urea PAGE, and full length transcripts were excised from the SYBR Gold stained gel (Thermo Fisher Scientific), eluted overnight in G50 buffer (20 mM Tris HCl pH 7.5, 300 mM NaOAc, 2 mM EDTA, 0.2% SDS), phenol/chloroform extracted, and ethanol . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 precipitated. The RNA (~40 pmol) was dephosphorylated using shrimp alkaline phosphatase (rSAP, NEB), labeled with 1 µl [ᵧ 32 P] ATP (150 mCi/ml) using USB Optikinase (Affymetrix), then gel purified as described above and dissolved in 20 µl of nuclease free water. For the in-line probing reaction, 1 µl RNA (≥ 20,000 cpm) was incubated in 2x reaction buffer (100 mM Tris-HCl pH 8.3, 40 mM MgCl 2 , 200 mM KCl) at room temperature for 24 h or 48 h. The reaction was quenched with 2x loading buffer (10 M urea, 1.5 mM EDTA pH 8.0). To generate ladders, 1 µl of the purified RNA was separately subjected to hydrolysis using the Next Magnesium RNA Fragmentation module (-OH) or RNase T1 digestion (T1) (NEB). Reactions were resolved by 8% Urea PAGE, exposed on a phoshorimager screen, and scanned using the Storm 820 imaging system (GE Healthcare). Deduced RNA structures were drawn using the RNA secondary structure visualization tool forna (Vienna RNA Web Services).