Figure 2. The effects of Top1 depletion on RNAPII regulation are linked to an underlying defect in ribosome biogenesis.

(A) Northern blot of 5’ETS1-containing rRNAs prepared from cultures of wild-type, Top1-AID, Top2-AID and Top1/2-AID cells that had been pulse-labeled for 2 min with [³H] adenine at the indicated times following addition of auxin to the media. Total RNAs were extracted and samples were separated on agarose gels, transferred to a nylon membrane and first directly autoradiographed to reveal pulse labeling of nascent rRNAs (see Figure 2—figure supplement 1C). The membrane was next hybridized with a ³²P-labeled oligonucleotide probe allowing detection of all species containing 5’ETS1 (ACGACAAGCCT-ACTCGAATTCGT). Truncated pre-rRNA fragments, first identified in cells lacking Top1 (El Hage et al., 2010), are indicated (*). (B) Polysome sedimentation profiles (OD₂₆₀) of WT, Top1-AID, Top2-AID, and Top1/2-AID strains 20 min following auxin treatment (large panels, as indicated). The top of each gradient (fractions 7 to 11), corresponding to 40S and 60S subunit peaks, is expanded below, where peak height differences (60S:40S ratio) are indicated. (C) Total cell extracts prepared from the indicated fractions of sedimentation profiles of WT and Top1/2-AID strains (from B) were TCA precipitated and analyzed by Western blot following SDS-PAGE, using an antibody against Rpl3 and Rps8, as indicated. (D) Total (left panels) and detergent-insoluble pellet (right panels) fractions isolated from lysates of Top1/2AID cells treated (+) or not (-) with auxin were analyzed by SDS-PAGE and Coomassie blue staining (top panels) or immunoblotting with the indicated antibodies (bottom panels). The pellet fraction is overloaded 25-fold compared to the total extract fractions. (E, F, G) Scatter plots (top panels) comparing RNAPII (Rpb1) ChIP-Seq read counts for individual genes in Utp8-AID (E) or Utp13-AID (F) cells after 20 min of auxin or vehicle treatment, or WT cells after 20 min of treatment with diazaborine or vehicle (G) (y-axis: auxin or diazaborine) for 20 min versus non-depleted cells (x-axis, Vehicle). Each dot represents a gene (5041 genes in total) and genes are color-coded according to functional groups, as in Figure 1A. Bottom panels display the corresponding box plots for the four indicated gene categories plus all other genes (others).

Figure 2—figure supplement 1. Protein localization and transcriptional effecs of Top1, Top2 and Top1/2 depletion.

(A) A Top1/2-AID strain expressing Btn2-eGFP and Nop1-mCherry was grown to exponential phase and cell samples were used for fluorescence microscopy analysis after 20 min of vehicle (Veh) treatment (mock depletion; top panels) or auxin (Aux) treatment (Top1/2 depletion; bottom panels). The number of Btn2-eGFP foci per cell was quantified and is presented in the panel to the right. 75–100 cells were quantified for each experiment, and the data are reported as averages from two experiments, with standard deviations indicated. (B) A Top1/2-AID strain expressing Pre6-eGFP and Nop56-mCherry was grown exponentially and cell samples were used for fluorescence microscopy analysis after 20 min of Top1/2 depletion by auxin treatment (Aux) or mock depletion (Veh). The number of cells where Pre6-eGFP showed a peri-nuclear localization was quantified and is presented as a percentage. 75–100 cells were quantified for each experiment, and the data are reported as averages from two experiments, with standard deviations indicated. (C) Yeast cells (either wild type, Top1-AID, Top2-AID or Top1/2-AID, as indicated) were grown synthetic glucose medium lacking adenine to mid-log phase at which point auxin was added (to 0.5 mM) and cells were then pulse labeled with [³H] adenine for 2 min at different time points after auxin addition (T = 0, 10, 20, 30 min). RNAs were extracted and samples were separated on agarose gels, transferred to a nylon membrane. After documenting ³H labeling by autoradiography, the same membrane was used for the Northern blot shown in Figure 2A. (D) RNAPII ChIP occupancy at the RPL30 and RPL39 promoters or at the SSA4 and HSP42 promoters at 20 min following Top1 and Top2 depletion.