Pre-rRNA Pulse Chase Assay Protocol

Pre-rRNA pulse chase assays were performed as previously described^{62 (link)}, with minor modifications. Briefly, 50 mL of cells were grown at 30 °C in EMM-ura to OD_600nm of ∼0.5–0.8. The cells were then diluted to an OD_600nm of 0.018 (WT) or 0.03 (Pnmt1-seb1) where 60 µM of thiamine was added for 15 h at 30 °C. Cells were resuspended in 850 μl of EMM-ura containing 125μCi of uridine-³H (Perkin Elmer, cat no: NET367250UC). Following a 4 min pulse, 200 μl of cells were diluted in 1.7 mL of EMM supplemented with uracil and 60 μM of thiamine. At various times point, cells were taken and rapidly frozen in liquid nitrogen. RNA was purified and separated as described in the ‘preparation and analysis of RNA’ section, with the following modifications: 10,000 cpm of radioactivity from each sample were loaded and migrated onto a 0.8% agarose-formaldehyde gel. Finally, a tritium screen (BAS-IP TR 2025 E Tritium Screen, 28956482, Cytiva) was added to the membrane and analyzed suing a typhoon trio instrument.