Analysis of ribosome biogenesis

Northern blotting was performed as previously described³⁷. The hybridization signals were analyzed by phosphorimaging using a Typhoon biomolecular imager and ImageQuant software ver. 2.0.0.6 (Cytiva)(GE Healthcare), ImageQuant TL ver. 8.2 (Cytiva) and ImageQuant ver. 5.2 (Molecular Dynamics). Image processing was performed with Canvas ver. X.898 (ACD Systems, Inc.) . For oligonucleotide probe sequences, see Supplementary Table ¹. Cytoplasmic ribosomes were analyzed using an established procedure³⁵ with a few modifications as follows. Cycloheximide (CHX) was added to the culture medium to 50 μg/ml immediately prior to cell harvesting, after which cells were trypsinized and pelleted in ice-cold RPMI containing 50 μg/ml CHX at 1500 × g for 4 min. Cells were resuspended in 1 ml ice-cold buffer A1 (20 mM Tris-HCl pH 7.4, 130 mM KCl, 10 mM MgCl₂, 50 μg/ml CHX), transferred to a microcentrifuge tube, and pelleted at 1150 × g for 2 min at 4 °C. Cells were then lysed in 200 μl buffer A1 containing 0.5% Igepal CA-630, 0.5% sodium deoxycholate, 2.5 mM dithiothreitol (DTT), 0.2 mg/ml heparin, and 80 U/ml RiboLock (Thermo Fisher) for 10 min with nutation at 4 °C. Cell lysate was cleared by centrifugation at 10,000 × g for 10 min at 4 °C. The supernatant was layered on top of a 15–50% (w/v) sucrose gradient in 10 mM Tris-HCl pH 7.4, 60 mM KCl, 10 mM MgCl₂, 1 mM DTT, 0.2 mg/ml heparin, and 0.01% Brij 35. The gradients were centrifuged at 160,000 × g for 200 min at 4 °C in a Beckman SW41Ti rotor. The gradients were fractionated by upward displacement with 69% (w/v) sucrose in a Beckman Fraction Recovery System. The A₂₅₄ was measured using a BioRad EM-1 UV cell and the data were analyzed with WinDaq ver. 3.76 (DATAQ Instruments, Inc.).