Balanced production of ribosome components is required for proper G1/S transition in Saccharomyces cerevisiae

Abstract

Cell cycle regulation is a very accurate process that ensures cell viability and the genomic integrity of daughter cells. A fundamental part of this regulation consists in the arrest of the cycle at particular points to ensure the completion of a previous event, to repair cellular damage, or to avoid progression in potentially risky situations. In this work, we demonstrate that a reduction in nucleotide levels or the depletion of RNA polymerase I or III subunits generates a cell cycle delay at the G1/S transition in Saccharomyces cerevisiae. This delay is concomitant with an imbalance between ribosomal RNAs and proteins which, among others, provokes an accumulation of free ribosomal protein L5. Consistently with a direct impact of free L5 on the G1/S transition, rrs1 mutants, which weaken the assembly of L5 and L11 on pre-60S ribosomal particles, enhance both the G1/S delay and the accumulation of free ribosomal protein L5. We propose the existence of a surveillance mechanism that couples the balanced production of yeast ribosomal components and cell cycle progression through the accumulation of free ribosomal proteins. This regulatory pathway resembles the p53-dependent nucleolar-stress checkpoint response described in human cells, which indicates that this is a general control strategy extended throughout eukaryotes.

Keywords: Cell Cycle; Free Ribosomal Proteins; G1/S Transition; RNA Polymerase I; RNA Polymerase II; RNA Polymerase III; Ribosomal RNA (rRNA); Ribosome Assembly; Ribosomes; Transcription.

FIGURE 1.

Asynchronous cultures accumulate in G₁ (START) under NTP-depleting drug treatment. A, asynchronously growing wild-type and imd2Δ cells were treated with 100 μg/ml 6AU or MPA. Samples were taken at different time points to analyze DNA content by flow cytometry and the proportion of unbudded cells by microscopy. B, a representative microscopic (100×) image shows wild-type cultured cells before and after a 6-h 6AU treatment. Average cell size was also measured by flow cytometry. C, an α-factor-synchronized culture at START was incubated for a 15-min period with or without 6AU. Cells were then released, and samples were taken at the indicated times for the DNA content analysis by flow cytometry. D, an exponentially growing asynchronous culture was treated with 6AU (100 μg/ml) for 2 h. After three washes, half the culture was incubated with the α-factor for synchronization at START. The percentages of unbudded and small-budded cells were scored.

FIGURE 2.

Cell cycle regulators actively mediate the G₁ delay promoted by NTP-depleting drugs. A, experimental design and flow cytometry analysis of the yeast cultures used in B. Cells were synchronized with α-factor for 135 min. For those treated with 6AU, the drug was added during the last 15 min of synchronization and was maintained after the α-factor wash and release. Samples were taken after 120 min of synchronization (ST) and after the α-factor release at the indicated times. B, the mRNA levels of the RNA pol II-transcribed genes ACT1, IMD2, and the G₁ cyclins CLN1 (empty, +6AU, and filled, −6AU, triangles), CLN2 (empty, +6AU, and filled, −6AU, squares), and CLN3 are shown. A.U., arbitrary units. C, a SIC1::MYC strain was synchronized, treated with 6AU, and released as described above. Extracts from equal amounts of cells were loaded in SDS-polyacrylamide gels and analyzed by Western blotting with an anti-Myc antibody. D, effect of the CLN3 overexpression on cell cycle progression and cell viability during 6AU treatment. Wild-type cells containing a TET-off:CLN3-expressing plasmid were incubated for 6 h in the presence of 6AU for the flow cytometry analysis. The same strain was incubated in SC-URA with or without doxycycline (Dox) and with or without 6AU for the growth test by serial dilutions. E, effect of SIC1 deletion on cell cycle progression and cell viability in the presence of 6AU. A sic1::KAN strain was incubated for 6 h in 6AU and cells analyzed by flow cytometry. The same strain was assayed in a growth test in the presence of increasing concentrations of 6AU.

FIGURE 3.

NTP-depleting drugs differentially affect the levels of the three RNA polymerase transcripts. Exponentially growing cells were treated with 100 μg/ml 6AU. Samples were taken at the indicated times, and the RNA levels of RNA pol I, pol II, and pol III transcribed genes were analyzed. A, Northern blot analysis of high molecular mass pre- and mature rRNAs. Specific probes were used to reveal the different pre- and mature rRNAs shown in each panel. Mature rRNA 18S was used as the loading control. Signal intensities of Northern blot analysis of the pre- and mature rRNAs presented in A were measured by phosphorimaging; values were normalized to those obtained for the wild-type control before 6AU addition and arbitrarily set at 1.0. B, Northern blot analysis of SUP56 pre-tRNA and mature 5S rRNA. The first precursor transcript of SUP56 is indicated (*). C, Northern blot analysis of RNA pol II transcribed RPL5, RPS3, and ADH1 mRNAs. Mature rRNA 18S was used as the loading control. D, quantification of the relevant RNA species shown in A, B, and C. 35S pre-rRNA and RPL5 mRNA were normalized against the 18S rRNA signal, whereas the first precursor of SUP56 (*) was normalized against 5S rRNA. All the data have been obtained in relation to the untreated cells levels (time 0 h) and are expressed as the average of at least three independent experiments ± S.D. (error bars).

FIGURE 4.

Depletion of selected RNA pol I or RNA pol III subunits reproduces NTP-depleting drug-mediated RNA imbalance and induces G₁ cell accumulation. A, doubling time progression of a strain with the gene of the RNA pol I subunit RPA43 under the control of a TET-off promoter plus/minus doxycycline (Dox.). B, Northern blot analysis of RNA pol I-transcribed 35S pre-rRNA and of the mRNAs of RNA pol II-transcribed genes ADH1 and RPL5 during Rpa43 depletion. C, flow cytometry analysis, at the indicated times, of cell cycle progression during Rpa43 depletion after treatment with 100 μg/ml 6AU. D, doubling time progression of a strain with the gene of the RNA pol III subunit RPC17 under the control of a TET-off promoter with or without doxycycline. E, Northern blot analysis, during Rpc17 depletion, of the RNA pol III-transcribed pre-tRNA SUP56 (the first precursor is indicated *) and 5S rRNA, the mRNAs of the RNA pol II-transcribed genes RPL5 and ADH1, and the RNA pol I-transcribed 35S pre-rRNA. F, flow cytometry analysis, at the indicated times, of cell cycle progression during Rpc17 depletion in cells treated with 100 μg/ml 6AU.

FIGURE 5.

NTP-depleting drugs induce the appearance of free L5 r-protein which correlates with the G₁ response. A, a proteasome-deficient cim3-1 strain was grown in SC-URA at 30 °C (permissive temperature) and harvested at an A₆₀₀ of 0.5 after α-factor synchronization and with or without 15 min of 100 μg/ml 6AU treatment. Cell extracts were prepared, and 10 A₂₆₀ of each extract was resolved in 7–50% sucrose gradients. A₂₅₄ was continuously measured. Sedimentation is from left to right; 40S, 60S, 80S, and polysome peaks are indicated. Fractions were collected from the gradients, and proteins were extracted from an equal volume of each fraction and analyzed by Western blotting with the indicated specific antibodies. B, asynchronously growing wild-type, rpl11AΔ, and rpl11BΔ cells were treated with or without 100 μg/ml 6AU. Samples were taken at different time points, and the DNA content was analyzed by flow cytometry. C, wild-type and dst1Δ asynchronous cultures were treated, or not, with 100 μg/ml 6AU. Samples were taken at different time points and the DNA content analyzed by flow cytometry.

FIGURE 6.

Mutations in Rrs1 exacerbate both free L5 accumulation and G₁ delay in the presence of NTP-depleting drugs. A, Rrs1 is required for the incorporation of the L11 r-protein and the L5–5S r-protein-rRNA complex into pre-60S ribosomal particles. B, isogenic wild-type and rrs1-84 strains were synchronized as described in Fig. 5A in the absence or presence of 100 μg/ml 6AU. Cell extracts were prepared, and a 10 A₂₆₀ of each extract was resolved in 7–50% sucrose gradients. A₂₅₄ was continuously measured. Sedimentation is from left to right. Free, 40S, 60S, 80S, and polysome fractions are indicated. Fractions were collected from the gradients, and proteins were extracted from equal volume of each fraction and analyzed by Western blotting with anti-L5 and anti-PGK antibodies. C, asynchronous growing cells were treated with 100 μg/ml 6AU, and samples were taken every 2 h to analyze DNA content by flow cytometry. D, growth test of mutant alleles rss1-1, rss1-84, and their isogenic wild-type strain is shown. Serial dilutions were performed on SC-URA plates with or without MPA.