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. 2019 Sep 9;47(20):10865–10880. doi: 10.1093/nar/gkz771

Figure 5.

Figure 5.

RNA is associated with the P54nrb-RNaseH1-ASO complex. (A) Treatment with RNase A disrupts P54-RNase H1 association. HeLa cells were transfected with HIS-LgBiT-P54nrb or with SmBiT-RNaseH1 and HIS-LgBiT-P54nrb together. The following day cell lysates were prepared. 1/2 of each lysate was treated with RNase A at 1 μg/ml for 30 min. Following RNase A treatment the P54nrb/RNase H1 complex purified using Ni-NTA beads. Following 3 washes, bound material was eluted with 200 mM imidazole. NanoGlo substrate was then added and NLuc luminescence assayed. Red bars control; blue bars +RNase A. (B) RNAP I/II inhibitors prevent formation of P54-RNase H1 complex. HeLa cells were transfected with LgBiT-P54nrb or with HIS-SmBiT-RNaseH1 and HIS-LgBiT-P54nrb together. The following day cells were treated for 6 h with RNAP inhibitors. Following treatment cell lysates were prepared and the P54nrb/RNase H1 complex purified and quantitated as above red bars, no inhibitor; blue bars, 1 μg/ml Actinomycin D; green bars, 1 μM triptolide; grey bars, 0.5 μM flavopiridol. (C) Treatment of cells with RNAP I inhibitor CX-5461 inhibits formation of P54nrb-RNaseH1-ASO complex. Experiment was carried out as in B except following plasmid transfection cells were treated overnight with 300 nM CX-5461. (D) Identification of P54/RNase H1 associated RNAs by RIP-Seq. HeLa cells were transfected with HIS-SmBiT-RNaseH1 or with HIS-SmBiT-RNaseH1 and LgBiT-P54nrb. The P54nrb/RNase H1 complex was isolated the following day and RNA extracted. Directional, strand-specific libraries were prepared for NEXT-Seq analysis. Genes represented by more than 5 times the number of reads in RNaseH1/P54nrb vs RNaseH1 only are shown.