Cells depleted of Pwp2 accumulate pre-rRNAs before a decrease in 18S rRNAs levels. (A) Schematic of pre-rRNA processing in yeast. The SSU processome is required for pre-18S biogenesis at pre-rRNA processing sites A0, A1, and A2 (bold text). Oligos C, B, E, A, and Y were used as previously described (Lee and Baserga, 1997). Defects in these processing steps lead to accumulation of the 35S and 23S pre-rRNAs and a reduction in the 27SA2 and 20S pre-rRNAs and 18S rRNA. (B) Northern blot indicating that pre-rRNAs accumulate beginning at 8 h of Pwp2 depletion. Yeast depleted (GAL::PWP2) or not (YPH499) of Pwp2 were grown in YPG and shifted into YPD media. RNA was Northern blotted for pre-rRNAs and the mature 25S and 18S rRNAs. (C) Quantification of the results in B. Ratio of the 25S to 18S rRNAs (25S/18S ratio) were analyzed and plotted by time in YPD (Hours DEX) for GAL::PWP2 and YPH499. Ratio of the 20S/25S and 35S/25S RNAs were analyzed and plotted by time in YPD (Hours DEX). (D) Metabolic labeling indicating pre-rRNA processing defects at 9 and 12 h of depletion. GAL::PWP2 and YPH499 were grown in SG-URA and shifted into SD-URA media for 9 or 12 h. Cells were labeled with [³H]uracil and then incubated with complete media. RNA was extracted and equal counts were analyzed.

Accumulation of unbudded cells in Pwp2 depletion is Cln3 independent. (A) Cell growth and percentage of unbudded cells was monitored in cln3Δ Pwp2-depeleted yeast. GAL::PWP2 cln3Δ, cln3Δ, GAL::PWP2, and the parent strain (YPH499) were grown in YPG media and then shifted into YPD. (B) Cln3 protein levels decrease after 14 h of Pwp2 depletion. Yeast with Cln3-HA-MYC tagged in a GAL:: 3xHA-PWP2 strain was grown in YPG media and then maintained in early log phase in YPG or shifted into YPD media. Protein was extracted from an equal number of cells and analyzed by Western blot for Cln3 (anti-MYC antibodies), Pwp2 (anti-HA antibodies), Mpp10 (anti-Mpp10 antibodies), and Adh1 (anti-Adh1 antibodies) protein expression.

Increased nuclear residence time of Whi5 when ribosome biogenesis is disrupted. Using time-lapse microscopy, individual mother and daughter Whi5-GFP cells were analyzed in GAL::PWP2 and PWP2 yeast from a series of three movies representing different preincubation times in dextrose (either 4, 5.5, or 7 h). Data from all three movies were plotted on the same graph. The number of total hours in dextrose (Pwp2 depletion) is plotted on the x-axis and individual cell cycle interval is plotted on the y-axis in minutes. The time limit for each movie is shown with a diagonal line representing the maximum time for an event that could be observed. Uncompleted events, represented as symbols on the diagonal line, are assigned to the movie limit line. Individual mother cells are represented in pink, and daughter cells are blue. (A) Cell cycle analysis of individual Whi5-GFP yeast can be divided into three parts: 1) Mother bud emergence to Whi5 nuclear entry (MBE-W5I), 2) Whi5 nuclear entry to exit (W5I-W5O), and 3) Whi5 nuclear exit to bud emergence (W5O-BE). (B) Graph of time interval indicating Whi5 nuclear residence (W5I-W5O) in GAL::PWP2 and PWP2 yeast, as a function of time after transfer to dextrose that Whi5 nuclear entry occurred. Still frame examples of yeast depleted or not of Pwp2 (9 and 14.5 h dextrose) with nuclear Whi5-GFP foci marked with white arrows are from the 5.5 h dextrose preincubation movie. (C) Graph of time interval indicating Whi5 nuclear exit to bud emergence (W5O-BE) in GAL::PWP2 and PWP2 yeast, as a function of time after transfer to dextrose that Whi5 nuclear exit occurred. (D) Graph of the time interval indicating mother bud emergence to Whi5 nuclear entry (MBE-W5I) in GAL::PWP2 and PWP2, as a function of time after transfer to dextrose that mother bud emergence occurred.

Yeast depleted of Pwp2 accumulate unbudded cells and bud at a larger cell size. (A) Protein levels of Pwp2 decrease by 4 h of depletion. GAL::3xHA-PWP2 or Pwp2-3xHA yeast were grown to early log phase in YPG and then shifted into YPD. Equal amounts of cells were collected, and protein was extracted and analyzed by Western blot for Pwp2 (anti-HA antibodies) or Adh1 (anti-Adh1 antibodies). (B) Cell growth, percentage of unbudded cells, and cell size were monitored in Pwp2-depeleted yeast. GAL::PWP2 and the parental strain (YPH499) were grown in YPG media and shifted into YPD media. The percentage of unbudded cells was averaged from four independent experiments, and the SD is indicated. (C) Elutriation of Pwp2-depeleted and nondepleted yeast. GAL::PWP2 and YPH499 were grown in YPG media and then shifted into YPD media for 12 h. Yeast were elutriated and collected as fractions. Fractions were analyzed for percentage of unbudded cells versus cell size, RNA units versus percentage of unbudded cells, RNA units versus cell size (fl), protein units versus percentage of unbudded cells, and protein units versus cell size (fl). (D) Pwp2-depeleted yeast have enlarged vacuoles. GAL::3xHA-PWP2 and YPH499 were grown in SC-G and then shifted into SC-D media for 21 h. Cells were stained with vacuole stain FM4-64 and visualized by DIC or for FM4-64 staining on a Zeiss Axioplan2 microscope.

A decrease in new protein synthesis is not detectable until 10 h of Pwp2 depletion. GAL::PWP2 and YPH499 were grown in medium containing galactose and raffinose as a carbon source and then shifted into medium containing glucose (DEX) for the indicated time periods. Cells were pulsed with Trans³⁵S-label, and TCA-precipitable protein was analyzed. ³⁵S-amino acid incorporation of GAL::PWP2 cells was normalized for each experiment to the same time point in the control strain, YPH499. The ratios of three independent sets of experiments were averaged, and the SD was calculated.

Cells with disrupted WHI5 delay G1 arrest upon Pwp2 depletion. (A) Cell growth, percentage of unbudded cells, and cell size were monitored in whi5Δ Pwp2-depeleted yeast. GAL::PWP2 whi5Δ and whi5Δ yeast were grown in YPG media and then shifted into YPD media. The same samples were also analyzed by FACS. The percentage of unbudded cells was averaged from three separate experiments, and the SD is indicated. The YPH499 and GAL::PWP2 averages are superimposed from Figure 1B. Three of the four experiments were conducted simultaneously with the whi5Δ and GAL::PWP2 whi5Δ strains. (B) Elutriation of whi5Δ Pwp2-depeleted yeast. GAL::PWP2 whi5 or whi5Δ were grown in YPG and then shifted into YPD for 12 h. Cells were elutriated and analyzed for percentage of unbudded cells versus cell size, RNA units versus percentage of unbudded cells, or protein units versus percentage of unbudded cells.

Timeline of cellular events that occur after Pwp2 depletion. 4 h, Pwp2 protein levels are reduced; 8 h, steady state pre-rRNAs accumulate, new ribosome synthesis is delayed, and unbudded cells accumulate; 10 h, new proteins synthesis begins to decrease; 12 h, ratio of 25S/18S increases, and translation and growth rates are significantly decreased; 14 h, Cln3 protein levels are reduced; and 21 h, cell size has substantially increased.