Reduction in ribosomal protein synthesis is sufficient to explain major effects on ribosome production after short-term TOR inactivation in Saccharomyces cerevisiae

Abstract

Ribosome synthesis depends on nutrient availability, sensed by the target of rapamycin (TOR) signaling pathway in eukaryotes. TOR inactivation affects ribosome biogenesis at the level of rRNA gene transcription, expression of ribosomal proteins (r-proteins) and biogenesis factors, preribosome processing, and transport. Here, we demonstrate that upon TOR inactivation, levels of newly synthesized ribosomal subunits drop drastically before the integrity of the RNA polymerase I apparatus is severely impaired but in good correlation with a sharp decrease in r-protein production. Inhibition of translation by cycloheximide mimics the rRNA maturation defect observed immediately after TOR inactivation. Both cycloheximide addition and the depletion of individual r-proteins also reproduce TOR-dependent nucleolar entrapment of specific ribosomal precursor complexes. We suggest that shortage of newly synthesized r-proteins after short-term TOR inactivation is sufficient to explain most of the observed effects on ribosome production.

FIG. 1.

TOR inactivation modestly affects production of 35S pre-rRNA and RNA Pol I association with rRNA genes but strongly impairs downstream rRNA maturation events. (A) Simplified scheme of rRNA processing in yeast. (B) Neosynthesized rRNA levels decrease upon rapamycin treatment. An exponentially growing yeast strain (y2180) was cultured in the absence (0) or presence (200 ng/ml) of rapamycin (RAP) for the times indicated above the panel. At different time points, pulse-labeling with [³H]uracil was performed for 15 min. RNA was isolated, separated in a denaturing agarose gel, and transferred to a positively charged membrane. Tritium-labeled RNA was visualized using a BAS1000 imager (Fujifilm) (upper panel). The same blot was subjected to Northern analysis with a ³²P-labeled probe hybridizing to the 25S rRNA sequence (lower panel). Positions of the different rRNA processing products are indicated on the right. (C) Cellular [³H]uracil uptake decreases upon rapamycin treatment. An exponentially growing yeast strain, y658, was cultured in the absence (YPD) or presence (200 ng/ml) of rapamycin for the times indicated below the x axis. At the different time points, pulse-labeling with [³H]uracil was performed for 5 min. The cell-associated tritium label was determined, normalized to the value at the start of the time course experiment, and plotted against the time. (D) The level of neosynthesized 35S rRNA is less affected than 20S rRNA and 27S rRNA production after rapamycin treatment. Quantitative analysis of the primary data presented in panel B is shown. The tritium signal of the respective precursor was normalized to the ³²P signal of the Northern analysis. The 35S/25S rRNA ratio at the start of the time course experiment was arbitrarily set to 1. (E) Rapamycin treatment leads to a strong rRNA maturation defect. Quantitative analysis of the primary data presented in panel B is shown. The ratio of the tritium signals of the 35S and 27S rRNA precursors was calculated for each time point. The 35S/27S rRNA ratio at the start of the time course experiment was arbitrarily set to 1. (F and G) RNA Pol I association with 35S rRNA genes and endogenous Rrn3 levels are unaltered after 15 min of rapamycin treatment. Yeast strain y658, expressing RPA43 and RRN3 as fusion proteins with C-terminal triple HA epitope and a TAP tag, respectively, was grown to exponential phase. The culture was split in two parts and either cultured in YPD medium or treated with rapamycin (200 ng/ml). After the times indicated (below the bar charts on the left or on top of the panels on the right), two samples were withdrawn. Cells were either treated with formaldehyde at 30°C for 15 min for subsequent ChIP experiments or collected for preparation of whole-cell extracts and Western blot analysis. ChIP was performed as described in Materials and Methods using an antibody directed against the HA epitope of Rpa43-HA. The data presented in the bar graphs on the left are the average of three independent ChIP experiments, each analyzed in triplicate quantitative PCRs. Primer pairs were specific for the 35S rRNA gene promoter (Prom), for the 18S and 25S rRNA coding sequences (18S and 25S), and for the 5S rRNA coding sequence (5S) as a control. The positions of the amplified rDNA regions are indicated in the cartoon on the bottom of panel G. The fraction of the total input DNA recovered after ChIP for the different regions and time course experiments is depicted. Panels on the right depict the result of the corresponding Western blot analysis using antibodies specific for the C-terminal tags of Rpa43-HA and Rrn3-TAP. The same amounts of whole-cell extract (10 μg) were loaded in each lane. The signal ratios of Rrn3-TAP to Rpa43-HA are given at the bottom of the respective pair of panels.

FIG. 2.

The integrity of the RNA Pol I transcription machinery bound to the 35S rRNA gene is not impaired after 15 min of rapamycin treatment. (A) Rpa43 and Hmo1 association with 35S rDNA is unaltered after 15 min of rapamycin treatment. Two exponentially growing yeast strains (y622 and y621), expressing either Rpa43-MNase or Hmo1-MNase fusion proteins from the respective chromosomal gene locus, were treated with rapamycin (200 ng/ml) for 15 min. Samples were collected before (0) and after (15) rapamycin addition. After formaldehyde cross-linking for 15 min at 30°C and harvesting of cells, crude nuclei were isolated. The nucleus suspension was incubated in the absence (0) or presence of calcium, activating DNA cleavage by MNase fusion proteins for the times indicated on top of the panels (min ChEC). DNA was isolated, linearized with the restriction endonuclease XcmI, separated in an agarose gel, and analyzed by Southern blotting by indirect end-labeling using an rDNA-specific probe. The cartoon on the right shows a map of the corresponding 4.9-kb XcmI rDNA fragment to localize the cleavage events mediated by the MNase fusion proteins. The positions of regulatory elements within the rRNA gene promoter, the upstream element (UE) and core element (CE), of the 18S, 5.8S, and 25S rRNA coding sequences and of the transcription start site (arrow) and the target sequence of the radioactive probe are depicted. An arrow on the left of the blot marks the position of the full-length XcmI fragment. (B) The ratio of actively transcribed to transcriptionally inactive rRNA genes does not decrease after 15 min of rapamycin treatment. Aliquots of nuclei from the experiment presented in panel A before (0) and after 30 min of ChEC were subjected to psoralen cross-linking. DNA was isolated, linearized with the restriction endonuclease EcoRI, separated in an agarose gel, and analyzed by Southern blotting with a probe recognizing 2.8-kb and 1.9-kb EcoRI fragments containing the 25S and 18S rRNA coding sequences, respectively. The positions of the different fragments are indicated on the left. The fraction of actively transcribed rRNA genes is represented by the heavily psoralen-cross-linked s-band, whereas the poorly psoralen-cross-linked f-band originates from the transcriptionally inactive nucleosomal rRNA genes (marked on the right). (C to D) RNA Pol I associated with rRNA genes after 15 min of rapamycin treatment is not stalled on the DNA template. Panel C shows a flow chart of the experiment presented in panel D. Two yeast strains (y943 and y936), both expressing Rpa43-MNase from the chromosomal gene locus and carrying either an RRN3 wild-type allele or the temperature-sensitive rrn3-8 allele were grown to exponential phase (OD₆₀₀ of 0.5) at 24°C in YPD medium. The cultures were split in two, and rapamycin (200 ng/ml) was added to one half of the cells. After 15 min samples were withdrawn from the rapamycin (RAP)-treated and untreated (YPD) culture. Cells were treated with formaldehyde for 15 min at 24°C, harvested, and further analyzed in ChEC experiments as described in the panel A. The remainder of the cultures were shifted to 37°C and incubated for another 90 min before cells were treated with formaldehyde for 15 min at 37°C, harvested, and further analyzed in ChEC experiments as described for panel A. (D) Autoradiogram of the experiment, as described for panel A.

FIG. 3.

Rapamycin and cycloheximide short-term treatment affect rRNA maturation and RNA Pol I association with 35S rRNA genes to a similar degree. (A to D) rRNA processing defects after TOR inactivation and inhibition of translation are virtually identical. Yeast strain y658 was cultured to exponential phase. The culture was split in three parts and either further cultured in YPD medium or treated with rapamycin (200 ng/ml; RAP), or cycloheximide (100 μg/ml; CHX) for 15 min. Pulse-labeling with [³H]uracil was performed for 5 min, followed by a chase with an excess of unlabeled uracil (final concentration, 1 mg/ml) for the times indicated above the panels. RNA was analyzed (A) as described in the legend to Fig. 1B except that the autoradiogram shown was obtained after exposure of a blot treated with EN3HANCE solution (PerkinElmer). Cellular [³H]uracil uptake (B) after pulse-labeling of cells grown in the absence (YPD) or presence of rapamycin (200 ng/ml) or cycloheximide (100 μg/ml) for 15 min was determined as described in the legend to Fig. 1C, except that the experiment was performed twice for untreated cells (0 min) and in triplicate for cells after treatment (15 min). Quantitative analysis of tritium signals (C) presented in panel A was performed prior to EN3HANCE treatment as described in the legend to Fig. 1E. Tritium incorporation in 25S rRNA (D) was determined by excision of the 25S rRNA bands from an identical blot and analysis by liquid scintillation counting. The values obtained were normalized to the value after a 5-min pulse (0) of the culture grown for 15 min in YPD medium and plotted against the time of the chase. (E) RNA Pol I association with 35S rRNA genes and endogenous Rrn3 levels are unaltered after 15 min of cycloheximide treatment. Yeast strain y658 was cultured in the absence (YPD) and presence (100 μg/ml) of cycloheximide. ChIP and Western blot analyses were performed as described in the legend of Fig. 1F and G.

FIG. 4.

The defect in ribosomal subunit synthesis upon TOR inactivation correlates with shutdown of r-protein production. (A to C) r-protein production is severely and specifically affected shortly after rapamycin treatment. Panel A shows a flow chart of the experiment presented in panel C. Two wild-type yeast strains (y6 and y207) were grown to exponential phase (OD₆₀₀ of 0.5) at 30°C in SCD medium lacking methionine and cysteine (SCD-Met-Cys). Each of the cultures was split in six parts, and rapamycin (200 ng/ml; RAP), or cycloheximide (0.1, 1, 10 or 100 μg/ml; CHX) was added, with one of the samples remaining untreated. After another 15 min at 30°C, pulse-labeling with [³⁵S]methionine-cysteine was performed for 5 min. (B) Protein was isolated, and relative ³⁵S incorporation was determined as described in Materials and Methods. Values were normalized to the value obtained for the untreated cells growing in SCD-Met-Cys medium. The average and standard deviation of two independent biological replicates of strain y6 are shown. (C) Protein from the different samples described above, as well from affinity-purified 80S ribosome (80S), was separated in a 16% polyacrylamide urea gel. Total protein was detected with Coomassie blue, whereas neosynthesized proteins were detected after autoradiography of the gel. The position of a subset of proteins whose neosynthesis is specifically inhibited in the presence of rapamycin is marked by a bar on the left side of the autoradiogram. This area is enlarged for lanes 1 and 2 and 8 to 12 in the lower panels. Two asterisks mark protein bands whose major components have been unambiguously identified as r-proteins by mass spectrometry (Table 2). The experiment shown is from an analysis of strain y6. Identical results were obtained for strain y207. (D and E) Neosynthesis of r-proteins correlates with production of mature rRNAs. [³H]uracil pulse-chase experiments (D) were performed as described in the legend to Fig. 3A. The autoradiogram for strain y207 is shown. An identical autoradiogram was obtained for strain y6. Relative tritium incorporation in 25S rRNA (E) was determined as described in the legend to Fig. 3D. The values obtained after a 5-min pulse followed by a 16-min chase were normalized to the value obtained for the culture grown for 15 min in YPD medium.

FIG. 5.

Cycloheximide treatment and depletion of individual r-proteins lead to nucleolar entrapment of Rrp12 and Nog1. (A) Yeast strains y1809 and y1807, expressing either Rrp12 or Nog1 as a fusion protein with a C-terminal GFP from their respective genomic locations, were grown at 30°C in YPD medium to exponential phase (OD₆₀₀ of 0.5). The cultures were split in three and further incubated in the absence (YPD) or presence of either rapamycin (200 ng/ml; RAP) or cycloheximide (100 μg/ml; CHX). After the time points indicated at the top of the panels, a sample was withdrawn, washed with SCD medium, and immediately analyzed by fluorescence microscopy (GFP). Yeast cell morphology was visualized in parallel by differential contrast (DIC) optics. (B) Yeast strains y1809, y1807, y1805, and y1804, expressing either Rrp12 or Nog1 as a fusion protein with a C-terminal GFP from their respective genomic loci and expressing the ribosomal proteins rpS5 or rpL25 either from their respective genomic locations (WT) or from a plasmid under the control of a galactose-dependent promoter (pGAL-RPS5 and pGAL-RPL25), were grown at 30°C in YPG medium to exponential phase (OD₆₀₀ of 0.5). The cultures were split in two, and cells were collected, washed, and further incubated in either YPG or YPD medium. After 90 min a sample was withdrawn, washed with SCD medium, and immediately analyzed by microscopy as described for panel A.