Coupling of RNA polymerase III assembly to cell cycle progression in Saccharomyces cerevisiae

Abstract

Assembly of the RNA polymerases in both yeast and humans is proposed to occur in the cytoplasm prior to their nuclear import. Our previous studies identified a cold-sensitive mutation, rpc128-1007, in the yeast gene encoding the second largest Pol III subunit, Rpc128. rpc128-1007 is associated with defective assembly of Pol III complex and, in consequence, decreased level of tRNA synthesis. Here, we show that rpc128-1007 mutant cells remain largely unbudded and larger than wild type cells. Flow cytometry revealed that most rpc128-1007 mutant cells have G1 DNA content, suggesting that this mutation causes pronounced cell cycle delay in the G1 phase. Increased expression of gene encoding Rbs1, the Pol III assembly/import factor, could counteract G1 arrest observed in the rpc128-1007 mutant and restore wild type morphology of mutant cells. Concomitantly, cells lacking Rbs1 show a mild delay in G1 phase exit, indicating that Rbs1 is required for timely cell cycle progression. Using the double rpc128-1007 maf1Δ mutant in which tRNA synthesis is recovered, we confirmed that the Pol III assembly defect associated with rpc128-1007 is a primary cause of cell cycle arrest. Together our results indicate that impairment of Pol III complex assembly is coupled to cell cycle inhibition in the G1 phase.

Keywords: G1 arrest; RNA polymerase III; Rbs1; assembly; cell-cycle; tRNA; transcription; yeast.

Figure 1.

Cell proliferation defects and morphological changes in the rpc128-1007 mutant can be partially corrected by RBS1 overexpression. Control strain (WT), isogenic rpc128-1007 mutant and rpc128-1007 transformed with a multicopy RBS1 plasmid, rpc128-1007 [RBS1], were grown in YPD medium at 30°C and harvested during the logarithmic (panels a-d) or stationary phase (panel c). (a) Morphology of rpc128-1007 mutant cells observed by phase microscopy. (b) Size distribution of cell populations measured using flow cytometry for forward angle scattering (FSC). (c) Percentage of unbudded cells among cells harvested during the logarithmic (log-phase) and stationary phase (stat-phase) was estimated after inspection of at least 100 cells. Bars present the mean value from three independent experiments with standard deviation. (d) Pheromone response assay using 750 µM α-factor. The zone of growth inhibition was measured and the respective values are presented in Table 1.

Figure 2.

rpc128-1007 inhibition of cell cycle progression at the G1 phase can be partially overcome by RBS1 overexpression. Flow cytometry histograms of cellular DNA content. The two prominent peaks reflect the 1C and 2C DNA content of cells in G1 and G2/M, respectively. The trough between the peaks reflects the relative number of cells in the S phase. (a) Flow cytometry histograms of wild type (WT), rpc128-1007 mutant and rpc128-1007 transformed with a multicopy RBS1 plasmid, rpc128-1007 [RBS1]. Asynchronous logarithmically growing cells (AS), after synchronization using α-factor (time point 0) and after transfer to fresh medium lacking α-factor at the indicated time points. The differences in the cell cycle are also visible in asynchronous cells. (b) Flow cytometry histograms of the TET:RPC17 mutant harboring the RPC17 gene under control of the TET-off promoter transformed with empty plasmid, TET:RPC17 [-] and RBS1 plasmid, TET:RPC17 [RBS1]. Cells were examined after addition of doxycycline (final concentration, 5μg/ml) to logarithmically growing cultures. A WT strain (BY4741) was used as a control (right panel).

Figure 3.

Deletion of the RBS1 gene causes mild delay in exit from the G1 phase. (a) Scheme of α-factor-nocodazole trap assay used and percentage of wild type (WT) and rbs1Δ cells remaining in G1 after release from α-factor synchronization as determined by this assay. Two cell populations, shmoo and budded, were observed by phase microscopy; cells showing shmoo were quantified. Bars represent the mean with standard deviation of three independent experiments. p values were calculated using Student’s t-test as ratios of WT to rbs1Δ cells in different time points after release from α-factor and showed statistical significance from 10 min to 65 min (p value <0.01). (b) Scheme of G1 cell synchronization in response to α-factor and percentage of cells in the G1 phase synchronized with α-factor for 90 min. Cells showing shmoo were quantified during this period. The mean with standard deviation was calculated from two independent experiments.

Figure 4.

MAF1 gene deletion elevates tRNA levels in rpc128-1007 cells. (a) Phenotype analysis of wild type (WT), rpc128-1007, maf1Δ and rpc128-1007 maf1Δ strains. Cells were incubated on YPD medium at 16°C or 30°C and on YPGly medium at 37°C. (b) Northern analysis of RNA using probes specific for tRNAs: t(L)CAA, t(Y)GUA and t(F)GAA.: tRNA precursor containing introns and unprocessed 5’, 3’-ends;: tRNA precursor with intron and unprocessed 3’-end;: end-matured intron-containing pre-tRNA;: mature tRNA. Cells were grown to logarithmic phase in YPD medium at 30°C and then shifted to 16°C for 2 h. (c) Amounts of primary transcripts () were normalized relative to the loading control (5,8S rRNA) and calculated relative to the amounts present in the WT strain, which were set to 1. Bars represent the mean with standard deviation of three independent experiments.

Figure 5.

Arrest of the double rpc128-1007 maf1Δ mutant in the G1 phase can be partially overcome by increased RBS1 expression. Wild type (WT), rpc128-1007 mutant, and double rpc128-1007 maf1Δ transformed with empty vectors [-] or multicopy [MAF1] and [RBS1] plasmids were grown to logarithmic phase in YPD medium at 30°C. (a) Flow cytometry histograms showing DNA content of asynchronous cells during logarithmic growth (AS), after synchronization by α-factor (time point 0) and after transfer to fresh medium lacking α-factor at the indicated time points. Note that differences in the cell cycle are also visible in logarithmically growing cells. (b) Number of asynchronous cells in the G1 phase and (c) after synchronization with α-factor and transfer to fresh medium at the indicated time points were calculated from three independent experiments. Bars in panel b and curves in panel c represent the mean with standard deviation.