![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 18, Issue 3, 953-964, March 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
,
,||
Departments of Molecular Biophysics and Biochemistry, *Genetics, and ||Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520; and Rockefeller University, New York, NY 10021
Submitted June 9, 2006;
Revised December 5, 2006;
Accepted December 20, 2006
Monitoring Editor: Karsten Weis
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Processing of the pre-18S rRNA occurs via a large complex of more than 40 nonribosomal proteins and the U3 snoRNA termed the small subunit (SSU) processome/90S pre-ribosome (Dragon et al., 2002
; Grandi et al., 2002; Schäfer et al., 2003). This complex can be visualized in chromatin spreads as the knobs on the ends of the nascently transcribed pre-rRNAs (Dragon et al., 2002; Gallagher et al., 2004). Now that many components required for making ribosomes have been identified, recent work has focused on understanding how ribosome biogenesis is regulated and connected to other cellular processes including the cell cycle.
Recent work has shown a connection between ribosome biogenesis and the cell cycle. In yeast, depletion of SSU processome proteins leads to G1 delay and an inability to reenter the cell cycle (Bernstein and Baserga, 2004); other cell cycle defects upon inhibition of ribosome biogenesis in yeast have been observed (Du and Stillman, 2002; Oeffinger and Tollervey, 2003; Jorgensen et al., 2004a; Saracino et al., 2004). In animal cells, removal of BOP1, which is required for pre-rRNA processing of the LSU pre-RNAs, leads to G1 arrest (Pestov et al., 2001a, 2001b; Strezoska et al., 2002). Still, the mechanisms coordinating ribosome biogenesis and cell cycle are largely unknown.
Cell growth and division are coordinated processes in which cells must reach a critical size before cell division can occur. Classical studies defined a critical cell size or "setpoint" required for progression at Start, the point at which cells commit to cell division during late G1 (Hartwell et al., 1974; Johnston et al., 1977). Similar phenomena exist in a wide variety of organisms including bacteria, fission yeast, and avian erythroblasts (Donachie, 1968; Hartwell et al., 1974; Johnston et al., 1977; Nurse and Thuriaux, 1977; Dolznig et al., 2004). Arrest at Start in yeast can be caused by nutrient deprivation, mating pheromones, or translation defects (Unger and Hartwell, 1976; Hartwell and Unger, 1977; Johnston et al., 1977). It is not known whether the critical cell size setpoint sensed corresponds to cell volume, protein content per cell, RNA content per cell, or rates of protein or ribosome synthesis (Jorgensen and Tyers, 2004).
Cln3, a G1 cyclin, functions by activating the SBF (Swi4-Swi6) and MBF (Mbp1-Swi6) complexes, which transcriptionally activate many genes involved in DNA synthesis and repair (Wijnen and Futcher, 1999). Cln3 may be important for cell size sensing at Start. Cln3 is highly unstable and may be a rate-limiting Start activator (Cross and Blake, 1993; Tyers et al., 1993; Polymenis and Schmidt, 1997). Therefore, if translation is inhibited or ribosome levels decrease, then insufficient Cln3 protein might be available for the cell size setpoint and Start progression.
Recently, Whi5, a functional equivalent to human tumor suppressor Rb, has been identified as a key regulator of the yeast Start checkpoint (Costanzo et al., 2004; de Bruin et al., 2004; Jorgensen and Tyers, 2004). Whi5 negatively regulates passage through Start by inhibiting SBF and MBF activation and may itself be inhibited by Cln3-Cdc28–dependent phosphorylation.
Regulation of expression of the ribosome biogenesis regulon (RiBi) and the ribosomal proteins themselves has recently been elucidated in yeast (Jorgensen et al., 2002) with the discovery of two transcription factors, Sfp1 and Sch9, which function in the Ras/PKA and TOR signaling pathways to control the ribosome biosynthetic rate (Fingerman et al., 2003; Jorgensen et al., 2004a; Marion et al., 2004). It has been proposed that expression of the RiBi regulon also affects the cell size setpoint for Start, because mutations that inhibit RiBi expression result in small cells passing Start (Jorgensen et al., 2002; Jorgensen and Tyers, 2004). The mechanism by which this occurs is unknown but is thought to be independent of the main Start regulatory pathway (Cln3, Whi5, SBF/MBF, Cln1/2).
Here we show that cells respond to deficiencies in ribosome biogenesis by delaying Start, expanding the early G1 part of the cell cycle during which Whi5 is nuclear. This delay occurs before a decrease in overall levels of ribosomes, total protein levels, or protein synthesis; thus, cells may anticipate mRNA translation deficiencies before the changes actually occur. As a result, the Whi5 protein is retained in the nucleus where it may inhibit passage through Start. When the WHI5 gene is disrupted, cells are unable to delay the cell cycle in response to changes in pre-rRNA levels, at least until overt ribosome depletion is detectable. In contrast, deletion of CLN3, the major Whi5 antagonist, has no effect on this mechanism. Our results suggest that ribosome biogenesis has a positive effect on Start and that when ribosome biogenesis is defective, Whi5 is retained in the nucleus. Combined with evidence that ribosome biogenesis may delay Start by increasing the cell size setpoint (Jorgensen et al., 2004), we conclude that ribosome biogenesis is sensed at multiple steps in Start regulation.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
63, his3-200, leu2-1). GAL-3xHA-PWP2 Whi5-GFP and Whi5-GFP yeast strains were derived from W303. Yeast was grown in rich medium YPD (1% yeast extract, 2% peptone, 2% dextrose), YPG/R (1% yeast extract, 2% peptone, 2% galactose, 2% raffinose), SC (synthetic complete) media with either -D (2% dextrose) or -G (2% galactose) and minus uracil (-URA) where specified. Yeast with C-terminal 3xHA-tagged proteins or N-terminal GAL-3xHA–tagged proteins were created (Longtine et al., 1998). Yeast expressing Cln3-HA-MYC (TRP marker) was genomically integrated with the pMM162 plasmid from Mary Miller (Miller et al., 2005). WHI5 and CLN3 disruptions were made using the CLONAT (pAG25) marker (Goldstein and McCusker, 1999).
Growth Curves
Cells were grown in YPG media to early log phase and then shifted into YPD media for 24 h. Cells were maintained in early log phase throughout the time course and analyzed for growth by measuring absorbance at OD600 or OD660; the percentage unbudded and DNA content were measured by flow cytometry on a FACSCalibur System (BD Biosciences, San Jose, CA) or cell size by Beckman Coulter counter Model Z2 (Fullerton, CA).
Vacuole Staining
YPH499 and GAL::3xHA-PWP2 were grown to early log phase in SC-G media. Cells were shifted into SC-D media for 21 h to an OD600 of 0.4. One milliliter of cells was gently sonicated, and 40 µm of FM4-64 (Molecular Probe T3166) was added. Cells were incubated for 1 h at 30°C, washed with SC-D, and fixed to a slide in 0.6% LMP agarose and visualized on a Zeiss Axioplan2 microscope (Thornwood, NY).
Time-Lapse Microscopy
Yeast strains expressing Whi5-GFP (Costanzo et al., 2004), PWP2 or GAL::3xHA-PWP2, were grown in SC-G media to early log phase, then shifted to SC-D media for 4, 5.5, or 7 h, and then analyzed by time-lapse microscopy as previously described (Bean et al., 2006). Fluorescent and phase-contrast images were acquired at 3 min resolution for 9 h. Only a subset of yeast was included in the analysis because of unscorable faint/undetectable Whi5-GFP expression or out-of-plane bud emergence.
Protein Analysis by Western Blot
Yeast with Pwp2-3xHA, GAL::3xHA-PWP2, or GAL::3xHA-PWP2/Cln3-HA- MYC were grown to early log phase in YPG. Cells were shifted into YPD or YPG media and maintained at an OD600 of 0.3–0.6. Equal numbers of cells were collected at either 0, 4, 8, 10, 12, 14, 16, 20, or 24 h of growth in YPG or YPD, and protein was extracted as previously described (Kushnirov, 2000). Protein was analyzed by 10% SDS-PAGE and Western blotted either with anti-HA antibodies (12CA5) to detect Pwp2, anti-Mpp10 antibodies to detect Mpp10 (Dunbar et al., 1997), anti-Myc antibodies to detect Cln3 (9E10) or anti-Adh1 antibodies (Chemicon, Temecula, CA; Ab1202) to detect the alcohol dehydrogenase protein (1:1500) as previously described (Bernstein and Baserga, 2004; Bernstein et al., 2004).
Cell Elutriation
One liter of YPH499, GAL-3xHA-PWP2, whi5, GAL-3xHA-PWP2 whi5 yeast were grown to early log phase in YPG/R media and then shifted into YPD media for 12 h. Yeast were sonicated in 75 ml of ice-cold water for 3 min total. Cells were elutriated (Beckman J-6M/E centrifuge, Fullerton, CA) at 4°C in cold water at 3000 RPM, and 12 fractions of 400 ml were collected and analyzed for percentage unbudded, DNA content, RNA content, and protein content as described below. Pump speeds varied by elutriation, but all were increased by 10% with each collected fraction.
RNA and Protein Analysis from Elutriated Fractions
For RNA analysis, 15 ml from each fraction was collected and washed two times with 1x PBS. Cells were resuspended in 0.5 ml Tris-HCl, 7.5 (200 mM)/NaCl (211 mM)/MgCl2 (78 mM), and 55 µl of propidium iodide (1 mg/ml). For protein analysis, equal numbers of cells from each fraction were collected and washed twice in 1x PBS. Cells were resuspended in 0.5 ml sodium bicarbonate (0.5 M) and 25 µl of fluorescein isothiocyanate (FITC; 1 g/l) were added, and cells were incubated on ice for 30 min. Cells were washed twice with 1x PBS, resuspended in 0.5 ml 1x PBS, and analyzed as described (Popolo et al., 1982).
RNA Analysis by Northern Blot
YPH499 or GAL::3xHA-PWP2 were grown to early log phase in YPG/R media and shifted into YPD media for up to 24 h. RNA was extracted from yeast from 25 ml of culture grown to an OD600 of 0.4 in YPD media for either 0, 4, 8, 12, 16, 20, or 24 h. Twenty micrograms of RNA was run on 1.25% agarose-formaldehyde gels as previously described (Lee and Baserga, 1999). To determine the 25S/18S ratios, 10 µg of RNA was run on a 1.25% agarose gel, stained with ethidium bromide, and analyzed using an Alphaimager 2200 (Alpha Innotech Corporation, San Leandro, CA). To quantitate the pre- rRNAs, the Northern blots were analyzed using a phosphoimager (Molecular Dynamics, Storm 840, Sunnyvale, CA).
Metabolic Labeling of RNA
YPH499 or GAL::3xHA-PWP2 containing pGAD3 uracil-based plasmid was grown to early log phase in SG/R-URA media for 48 h and shifted into SD-URA media for 9 or 12 h. Forty milliliters of cells at OD600 0.4–0.5 was then pulsed for 2 min with 100 µCi of [3H]uracil. RNA was extracted, and 20,000 cpm was analyzed as previously described (Dunbar et al., 1997).
Metabolic Labeling of Proteins
For quantitative analysis of new protein synthesis, YPH499 and GAL::3xHA- PWP2 cells were grown in SG/R-MET medium and, except for the 0-h time point, shifted into SD-MET medium for 8, 10, 12, 16, and 24 h. Similar to growth in rich medium, growth of Pwp2-depeleted cells was not affected until 12 h after the switch into dextrose-containing medium. We verified that the percentage of unbudded cells was already markedly increased by 8 h of Pwp2 depletion (data not shown). For metabolic labeling of proteins, 1 ml of cells of an OD600 0.5 was pelleted and resuspended in 1 ml SD-MET (8-, 10-, 12-, 16-, and 24-h time points) or SG/R-MET (0-h time point). After incubation at 30°C for 10 min, 3 µCi of Tran35S-label (MP Biomedicals, Solon, OH; 1175 Ci/mmol, 10.5 mCi/ml) were added. After 5 min labeling at room temperature, 400 µl of ice-cold chase buffer (21 mM cold methionine, 32% trichloroacetic acid [TCA]) was added, and cells were incubated at 65°C for 20 min. Cells were collected on GF/C filter paper using a Millipore (Bedford, MA) 1225 Sampling Manifold, washed three times with 5 ml ice-cold 5% TCA, and then washed three times with ice-cold 100% ethanol. GF/C filter papers were dried at room temperature, and the 35S incorporation was measured in a scintillation counter.
Flow Cytometry
Two milliliters of cells from YPH499, GAL::3xHA-PWP2, whi5, and whi5 GAL-3xHA-PWP2 were collected from early log phase cultures of yeast strains grown in YPG/R and shifted into YPD for 0, 3, 4, 6, 8, 9, 12, or 24 h. Samples were prepared and analyzed as previously described (Burton and Solomon, 2000).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). To examine this phenomenon further, we chose to study Pwp2/Utp1 as a representative of SSU processome components (Dragon et al., 2002; Dosil and Bustelo, 2004; Gallagher et al., 2004). For simplicity, we will refer to Pwp2/Utp1 by its original name, Pwp2. To deplete cells of Pwp2, we used a previously characterized strain (GAL::3xHA-PWP2) where Pwp2 is under control of a galactose-inducible (expressed; YPG/R) or dextrose-repressible (repressed; YPD) promoter. Western blot analysis demonstrated greatly reduced Pwp2 levels after 4 h in dextrose that became undetectable by 8–12 h (Figure 1A). In contrast, yeast where Pwp2 has only an epitope 3xHA tag (Pwp2-3xHA) did not reduce Pwp2 levels in dextrose. Similarly, levels of Adh1, an unrelated abundant protein, were not affected upon Pwp2 depletion (Figure 1A).
|
), cells were also analyzed for accumulation of unbudded cells, a different assay for G1 delay. Cells depleted of Pwp2 start to accumulate unbudded cells as early as 8 h after the switch to dextrose, indicating a delay in G1 (Figure 1B). Therefore, yeast depleted of Pwp2 have reduced Pwp2 protein levels at 4 h (Figure 1A), accumulated unbudded cells at 8 h but only significantly slow growth by 12 h in dextrose (Figure 1B; summarized in Figure 7). Throughout our experiments, we compared the effects of Pwp2 depletion in the GAL::PWP2 strain to the parent strain, YPH499, both grown in dextrose, and not to GAL::PWP2 nondepleted yeast grown in galactose. This is because it is most accurate to compare yeast grown in the same carbon source as it influences cellular growth rate, the percentage of unbudded cells, and cell size.
Previous work suggested that blocking ribosome biogenesis should lower the cell size setpoint for Start, resulting in passage of Start at smaller cell volumes (Jorgensen et al., 2004a). Therefore, we asked whether cells depleted of Pwp2 were altered in size. We directly measured cell volume using a Coulter counter over the same time period in dextrose (Figure 1B). Surprisingly, when measured at 24 h of depletion, cells depleted of Pwp2 were larger than wild-type cells (Figure 1B).
Cells Depleted of Pwp2 Bud at Larger Cell Size than Nondepleted Yeast with Moderately Reduced Cellular RNA Content
We asked whether the increase in cell size observed entails a parallel increase in cellular RNA and protein content. To do this, we analyzed yeast RNA and protein content per cell volume when Pwp2 is depleted. Cell cultures were fractionated by elutriation, a method that creates a gradient of cells that are collected as fractions based on size. The smallest cells often correspond to unbudded G1 cells and are collected in the first fractions. The larger cells frequently correspond to budded cells and are collected in subsequent fractions. Thus, yeast of similar sizes and budding indices can be compared for RNA or protein content per cell volume.
Cell elutriation was used to sort yeast depleted of Pwp2 for 12 h, because it is the time at which growth is significantly slowed (Figure 1C). We confirmed that GAL::PWP2- depeleted yeast were larger (Figure 1B) by analyzing elutriated fractions with cells at a budding index of 50%, which was used here as a reference point. At this budding index, GAL::PWP2 cells had a modal cell volume of 50 fl, whereas the parent cells (YPH499) had a volume of 34 fl (Figure 1C; Supplementary Table 1). These results suggest that Pwp2-depeleted yeast may have a larger critical volume for budding than wild type.
Elutriated yeast depleted or not of Pwp2 were analyzed for total RNA and protein content per cell volume. Total RNA was determined by single-cell assay using propidium iodide staining followed by FACS (see Materials and Methods). Protein content was determined by single-cell assay using FITC staining followed by FACS (Popolo et al., 1982). To compare the results between cells depleted or not of Pwp2, we used a reference point of an elutriated fraction with 50% budded cells. Cells depleted of Pwp2 (50 fl) had a mode value of 441 RNA and 477 protein units (arbitrary units), whereas nondepleted cells (34 fl) had a mode value of 592 RNA and 421 protein units (Figure 1C; Supplementary Table 1). These results indicate that although cells depleted of Pwp2 are larger by volume according to the Coulter counter, they have less RNA content than nondepleted yeast.
Yeast Depleted of Pwp2 Have Enlarged Vacuoles
One explanation for the increased volume of GAL::PWP2 cells without an increase in protein or RNA content could be an increase in the size of the vacuole. Yeast depleted or not of Pwp2 for 21 h were analyzed by differential interference contrast (DIC) microscopy and with the vacuole stain, FM4-64, which intercalates into the membrane of the vacuole (Figure 1D). Microscopy indicated that yeast depleted of Pwp2 had evidently larger vacuoles than nondepleted cells (Figure 1D).
Cells Depleted of Pwp2 Accumulate pre-rRNAs before a Decrease in the Levels of 18S rRNA
Pwp2 was previously found to be required for pre-rRNA processing of precursors to the 18S rRNA (Dragon et al., 2002; Dosil and Bustelo, 2004; Gallagher et al., 2004). We wanted to determine if the resultant pre-rRNA processing defects parallel the cell cycle changes we observed. Northern blot analysis of pre-rRNAs from cells depleted of Pwp2 showed that Pwp2-depeleted yeast accumulated the 35S and 23S pre-rRNAs and decreased the 27SA2 and 20S pre-rRNAs beginning at 
8 h after depletion (Figure 2, A and B; summarized in Figure 7). This effect was more pronounced after 12 h of depletion, when the growth rate was significantly decreased (Figure 2B; summarized in Figure 7). Because the levels of the 25S rRNA are not affected by Pwp2 depletion (Dragon et al., 2002; Dosil and Bustelo, 2004), we compared the ratio of the levels of the 35S and 20S pre-rRNAs to the 25S rRNA throughout the time course. These effects are quantitated in Figure 2C, where the 20S/25S ratio and the 35S/25S ratio clearly show that the 20S pre-rRNA decreases and 35S pre-rRNA accumulates beginning no later than 8 h after the shift to dextrose medium. Because pre-rRNAs began to accumulate, indicating defects in ribosome biogenesis, after 8 h of Pwp2 depletion, we asked whether levels of the mature 25S and 18S rRNAs would also change after 8 h. When Pwp2 was depleted, the ratio of 25S/18S rRNA increased after 12 h, indicating reduced 18S levels, which corresponded to the time when growth was significantly slowed (Figure 2C; see also Figure 7). In contrast, in nondepleted yeast grown in the same medium the ratio of 25S/18S rRNAs was approximately 1:1 throughout the time course (Figure 2C). Similarly, pre-rRNA processing defects were observed before changes in the ratio of 25S/18S rRNAs in Pwp2-depeleted cells by others (Dosil and Bustelo, 2004). Therefore, the observed increase in unbudded cells indicating G1 delay at 8 h occurs before the steady state levels of the mature 18S rRNA is decreased and thus may be directly related to defects in new ribosome synthesis that can be observed at the same time.
|
Because levels of the 18S rRNA are decreased by Pwp2 depletion, resulting in a decrease in the number of functional ribosomes, we asked whether new protein synthesis would also be affected after the shift to dextrose medium. GAL::PWP2 and YPH499 yeast were analyzed for new protein synthesis by metabolic labeling with Trans35S-label after growth in dextrose. TCA-precipitable proteins were collected on GF/C filter papers and the 35S-amino acid incorporation measured in a scintillation counter. The results show that at 8 h of Pwp2 depletion new protein synthesis is not yet reduced (Figure 3). By 10 h after the switch into dextrose-containing medium, new protein synthesis was slightly reduced and thereafter steadily decreased with progressive time of Pwp2 depletion. Thus, at 8 h of Pwp2 depletion, when unbudded cells are already accumulated and pre-rRNA processing defect become apparent, protein synthesis is not yet decreased (Figure 3).
|
; Cross and Blake, 1993) and therefore Cln3 translation would be sensitive to changes in ribosome levels, we asked whether the increase in unbudded cells from Pwp2 depletion is a result of decreased Cln3 protein levels.
We disrupted the nonessential CLN3 and asked if cln3 yeast also depleted of Pwp2 would accumulate unbudded cells. Pwp2-depeleted yeast and the parent strain YPH499 in a cln3 or CLN3 background were shifted into dextrose media and monitored for growth. After 12 h of Pwp2 depletion, cln3 and YPH499 yeast exhibited comparably slowed growth based on OD600 (Figure 4A). Because Pwp2-depleted yeast accumulated G1 cells, we asked if cln3 Pwp2-depeleted yeast would behave similarly. The results showed that cln3 yeast accumulated unbudded cells with kinetics similar to that of CLN3 yeast when Pwp2 was depleted, starting after 8 h in dextrose (Figure 4A).
|
). Correct tagging of Cln3 was verified by Western blotting with anti-Myc antibody. A single detectable Cln3 protein band ran at 65 kDa, comigrating with a protein band in a previously characterized tagged Cln3 strain (Miller et al., 2005). A band of that size was not detected in the untagged parent strain (YPH499; data not shown). Yeast depleted or not of Pwp2 were tested for Cln3, Pwp2, Mpp10, and Adh1 protein levels. As expected, Pwp2 protein levels were reduced after 4 h of depletion (Figure 4B). Cln3 protein levels, however, decreased only after 14 h of Pwp2 depletion (Figure 4B). Protein levels of Mpp10 (another known SSU processome component) and Adh1 (an unrelated abundant protein) did not change during this time course when Pwp2 was depleted (Figure 4B). Protein levels of the Cln3, Pwp2, Mpp10, and Adh1 proteins were unaffected when grown in YPG media (Figure 4B). Clearly, both the increase in unbudded cells and the slowing of growth precede the decrease in Cln3 protein levels. Therefore, consistent with our genetic results, a decrease in the levels of the Cln3 protein cannot be responsible for the G1 delay when Pwp2 is depleted.
Deletion of whi5 Blunts the G1 Delay Observed in Pwp2 Depleted Yeast
One candidate for mediating the G1 delay in response to inhibition of ribosome biogenesis is the Whi5 protein, because it is a known negative regulator of Start (Jorgensen et al., 2002; Costanzo et al., 2004; de Bruin et al., 2004). Therefore, we asked whether yeast with disrupted WHI5 would accumulate unbudded cells when ribosome biogenesis is inhibited. Yeast with GAL::PWP2 whi5 or whi5 were shifted into dextrose media (Figure 5A). After 12 h in dextrose, yeast depleted of Pwp2 (GAL::PWP2 whi5) slowed growth in comparison to whi5 (Figure 5A). Yeast were analyzed for changes in the cell cycle by 1) determining the percentage of unbudded cells and 2) analyzing cells by FACS. The results indicate that, in contrast to yeast where WHI5 is intact, yeast depleted of Pwp2 when WHI5 was disrupted did not accumulate unbudded or G1 cells with the same time course as yeast depleted of Pwp2 where WHI5 was intact (Figure 5A). However, after extended Pwp2 depletion, whi5 yeast did accumulate unbudded cells, but to a limited extent (Figure 5A). Therefore, disrupting WHI5 delays the G1 accumulation that is normally observed with Pwp2 depletion. Whi5 is thus likely involved in the initial cell cycle response, here examined at 8 h, to ribosome biogenesis defects. However, at later time points (24 h) when ribosome and protein levels are depleted and when protein synthesis is affected, a Whi5-independent mechanism likely ultimately causes G1 delay.
|
yeast were larger than whi5. In contrast to Pwp2-depeleted yeast, whi5 yeast did not significantly increase in cell size when Pwp2 was depleted for 24 h (Figure 5A). As expected from previously published results, whi5 yeast were smaller than the parent strain, YPH499 (Figure 5A and Supplementary Table 1; Jorgensen et al., 2002; Costanzo et al., 2004; de Bruin et al., 2004).
Because yeast depleted of Pwp2 have less RNA per cell volume, we asked if GAL::PWP2 whi5 yeast would also have less RNA per cell volume when compared with whi5. Cell elutriation was used to sort yeast by size depleted or not of Pwp2 for 12 h in a whi5 strain (Figure 5B). Yeast at the same budding index were compared for RNA or protein content per cell volume. The results indicate that GAL::PWP2 whi5 yeast incubated in dextrose were larger than whi5 yeast at a budding index of 50% (Figure 5B). GAL::PWP2 whi5 yeast had a mode value volume of 42 fl, whereas whi5 cells had a mode value volume of 29 fl (Figure 5B; Supplementary Table 1). Therefore Pwp2-depeleted cells were indeed larger than nondepleted cells when compared at the same budding index. These results suggest that although the accumulation of unbudded cells is delayed when WHI5 is disrupted and Pwp2 depleted, WHI5 disruption does not change the effects on cell size.
GAL::PWP2 whi5 yeast elutriated after incubation in dextrose for 12 h were analyzed for total RNA and protein content per cell volume as described previously for GAL:: PWP2 yeast. An elutriated fraction with 50% budded cells was used as a reference point. The results indicate that GAL::PWP2 whi5 yeast had a mode value of 367 RNA and 253 protein units, whereas whi5 cells had a mode value of 344 RNA and 258 protein units (Figure 5B). The results reveal that although GAL::PWP2 whi5 yeast are larger, they have roughly the same RNA and protein content when compared at the same budding index to whi5 yeast (Figure 5B; Supplementary Table 1).
Whi5-GFP Increases Time Spent in the Nucleus in Pwp2-depleted Cells
Previously, it was determined that Whi5 can inhibit SBF/MBF while in the nucleus (Jorgensen et al., 2002; Costanzo et al., 2004; de Bruin et al., 2004). To test whether deficiencies in ribosome biogenesis result in an increased interval of nuclear Whi5, expression of the Whi5-GFP protein was monitored in Pwp2-depeleted (GAL::PWP2) and nondepleted (parent strain; W303) yeast by time-lapse microscopy (Bean et al., 2006). By time-lapse microscopy of WHI5-GFP cells, growth of the mother cell (the cell before budding) and daughter cell (the bud on the mother cell) can be divided into three measurable intervals: 1) time from mother bud emergence to Whi5 nuclear entry (MBE-W5I), 2) time from Whi5 nuclear entry to Whi5 nuclear exit (W5I–W5O), and 3) time from Whi5 nuclear exit to bud emergence (W5O–BE; Figure 6A; Bean et al., 2006). It is important to note that because we were unable to construct the Whi5-GFP strain in the YPH499 strain background, the time-lapse microscopy was conducted using the previously characterized Whi5-GFP strain in a W303 strain background (Bean et al., 2006). W303 grows faster than the YPH499 strain background, thus altering the timing, but not the effects, of Pwp2 related cellular events (Supplementary Figure 1). The cell cycle delay observed with Pwp2 depletion due to whi5 was also seen in W303, although the effect was less pronounced, perhaps because of the faster growth rate in W303 (data not shown).
|
6 h in dextrose (Figure 6B). In addition, by the completion of the movies, many cells still had nuclear Whi5 (indicated by points lying on the diagonal lines). Still images from the GAL::PWP2 and PWP2 movies (9 and 14.5 h dextrose) are shown with nuclear Whi5-GFP foci marked with white arrows as an example (Figure 6B). Whi5 nuclear entry-to-exit time was longer in wild-type daughter cells than in wild-type mother cells, as previously observed (Bean et al., 2006). In Pwp2-depeleted yeast, consistent with our genetic results, increases in nuclear residence time of Whi5 occurs before the overt growth rate decreases (arrow in Figure 6B and Supplementary Figure 1).
Because Pwp2 depletion increases Whi5 nuclear retention time, we asked whether the time of bud emergence after Whi5 nuclear exit (W5O-BE) would also increase (Figure 6C). As previously observed (Figure 6C; Bean et al., 2006), budding followed Whi5-GFP nuclear exit by 25–30 min in PWP2 yeast (Figure 6C). Pwp2-depleted yeast did not significantly change this interval when compared with PWP2 yeast, at least until late in the depletion time course, significantly after clearly detectable increases in the Whi5 nuclear residence time (Figure 6C).
The range of time spent from mother bud emergence to Whi5 entry was similar for both GAL::PWP2 and PWP2 yeast, although some delay was detectable in the depleted cells late in the time course. Whi5 nuclear entry was simultaneous in both mother and daughter cells because of the sharing of common cytoplasm until mitotic exit (Figure 6D; Costanzo et al., 2004; Bean et al., 2006). In GAL::PWP2 and PWP2 cells, the interval between Whi5-GFP nuclear exit and completion of mitosis, as indicated by Whi5-GFP nuclear reentry required 40–120 min (Costanzo et al., 2004; Bean et al., 2006). These results indicate that upon inhibition of ribosome biogenesis, Whi5 nuclear residence time increases dramatically, before overt changes in cell growth rate, whereas other cell cycle intervals remain relatively unchanged until considerably later in the depletion time course.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To disrupt ribosome biogenesis we genetically depleted yeast of one protein that is required for SSU synthesis, Pwp2 (Dragon et al., 2002; Dosil and Bustelo, 2004; Gallagher et al., 2004), and monitored them for alterations (summarized in Figure 7). After 4 h, Pwp2 protein levels are visibly reduced (Figure 1A). After 8 h, defects in new ribosome synthesis (Figure 2, B–D), and an increase in unbudded cells (indicating G1 delay; Figure 1B) is observed. By 12 h of Pwp2 depletion, the growth rate has decreased (Figure 1B), translation rates are reduced (Figure 3), and the ratio of 25S/18S rRNAs is increased (indicating decreases in SSU; Figure 2C). By 14 h, Cln3 levels are decreased (Figure 4). By 21 h, cell size has substantially increased (Figure 1D). Whi5 retention in the nucleus accompanies increases in unbudded cells and pre-rRNA processing defects in the W303 strain (Figure 6B; Supplementary Figure 1). Because defects in cell cycle progression (Whi5 nuclear retention in W303 and increases in the percentage of unbudded cells) occur before a decrease in the number of functional ribosomes and translation, yeast may be sensing accumulation of 18S rRNA precursors or decreases in newly synthesized SSU subunits. Therefore, defects in ribosome biogenesis, and not decreases in the steady state number of functional ribosomal subunits, may be initially sensed at Start. Alternatively, yeast could be sensing a balance between new 60S and 40S subunit synthesis or decreases in new 40S subunit synthesis. In addition, we cannot exclude the possibility that yeast sense small changes in translation that are undetectable with our assays because changes in translation can clearly influence Start (Unger and Hartwell, 1976; Moore, 1988).
|
, 2004a). Yeast sense nutrients and stress via the Ras/PKA and TOR signaling pathways which signal to Sfp1 and Sch9 to alter synthesis of RiBi and RP regulons (Jorgensen et al., 2004a; Marion et al., 2004). Jorgensen et al. hypothesized that adequate nutrients influence Start at the level of ribosome biogenesis rather than by alterations in the protein synthesis rate (Jorgensen and Tyers, 2004; Jorgensen et al., 2004b). The mechanism of how Sfp1 and Sch9 couple ribosome biogenesis to Start through the SBF/MBF complexes is unknown, but it is thought that decreasing the rate of ribosome biogenesis lowers the critical size setpoint needed for budding. It has been hypothesized that these effects are independent of Cln3 and Whi5 because the cell size setpoint can be changed independently of CLN3/WHI5 (Jorgensen et al., 2004a).
Deficiencies in ribosome biogenesis in many eukaryotes have previously been shown to result in smaller cells (Shima et al., 1998; Montagne et al., 1999; Jorgensen et al., 2002, 2004a; Oliver et al., 2004; Ruvinsky et al., 2005). These results suggest that limiting ribosome biogenesis causes cells to set the cell size setpoint at a smaller cell volume. Although Jorgensen et al. analyzed the effects of alterations in transcription factors that globally regulate many downstream targets, we examined this phenomenon by inhibiting ribosome biogenesis through genetic depletion of the SSU processome and therefore analyzed events that happen downstream of Sfp1 and Sch9. In contrast to Jorgensen et al. (2004a), we found that inhibiting ribosome biogenesis (by Pwp2 depletion) did not result in smaller cell volumes. Furthermore, yeast depleted of Utp10 (another SSU processome protein), Fap7 (a protein required for cytoplasmic 20S pre-rRNA processing), and Rpf1 (a protein required for LSU biogenesis) also resulted in larger cell volumes (data not shown). Our results suggest that the cell cycle defects that we observe after 12 h are not related to reduced growth in cell volume because the volume of our cells is larger and suggests that yeast may be sensing cell "size" through another parameter (or parameters) such as RNA content, protein content, cytoplasmic volume, or the ratio of cytoplasmic to nuclear volume. This is important as the mechanism by which yeast sense "size" for cell cycle progression has remained unclear (Jorgensen and Tyers, 2004).
One explanation for the differences between our results and those of Jorgensen et al. could be that we analyzed the effects of depleting essential proteins instead of disrupting nonessential genes (Shima et al., 1998; Montagne et al., 1999; Jorgensen et al., 2002, 2004a; Oliver et al., 2004; Ruvinsky et al., 2005). Alternatively, we may have inhibited steps in ribosome biogenesis downstream of Sfp1 and Sch9, bypassing the mechanism that resets the cell size setpoint.
Although ribosome biogenesis may negatively regulate Start by increasing the cell size setpoint (Jorgensen et al., 2002, 2004a), we found that ribosome biogenesis can also positively promote Start. Our results suggest that cells can independently sense alterations in rates of ribosome biogenesis at Start before overt decreases in ribosome number or protein synthesis. We found that adequate ribosome biogenesis is needed to promote Start and that this effect is mediated, at least in part, by Whi5. Thus ribosome biogenesis may positively regulate Start by somehow inhibiting Whi5. Together these results suggest that ribosome biogenesis can have negative and positive regulatory roles on Start.
When we investigated why inhibition of ribosome biogenesis led to larger cells, we found that GAL::PWP2 yeast had enlarged vacuoles. Vacuole enlargement can have a major impact on cell volume, and indeed, at least some of the increased cell size in cln3 null cells (Cross, 1988; Nash et al., 1988) is due to vacuolar enlargement (Han et al., 2003, 2005). Vacuoles are analogous to mammalian lysosomes and function as a repository for metabolites and low-molecular-weight compounds. The enlarged vacuoles observed with Pwp2 depletion may be a result of cells undergoing autophagy or "self-eating" (Klionsky, 2005).
Because both Whi5 and Cln3 are important regulators of passage through Start (Jorgensen and Tyers, 2004; Jorgensen et al., 2004b), we hypothesized that these proteins might be mediating the cell cycle effects observed upon inhibition of ribosome biogenesis. Our results indicate that when ribosome biogenesis is inhibited by Pwp2 depletion, the Whi5 protein becomes retained in the nucleus, where it might contribute to delay of Start. Consistently, the G1 delay observed with Pwp2 depletion is delayed by whi5 deletion. Therefore, the initial response to ribosome biogenesis defects may occur through a Whi5-dependent mechanism. Alternatively, increased nuclear Whi5 could be a result, and not the cause, of the cell cycle defects observed. In any case, the genetic results that Whi5 is required for the cell cycle response (Figure 5) indicated that Whi5 is involved at some level. Although one role of Cln3 is to phosphorylate Whi5 to regulate its subcellular localization, our results suggest that defects in ribosome biogenesis leading to G1 delay occur independently of Cln3 protein levels. However, we cannot rule out the possibility that undetectably small changes in Cln3 protein levels or its modifications can result from Pwp2 depletion at earlier time points. Collectively, our genetic results suggest that the cell cycle defects observed with Pwp2 depletion were independent of Cln3 levels and dependent on Whi5. The genetic data were further supported by the observation that decreases in Cln3 protein levels occur both after the cell cycle defects and alterations in the cellular localization of Whi5 were observed. Therefore, there may be another labile cell cycle regulator that negatively regulates Whi5 in response to defects in ribosome biogenesis.
Our findings suggest that Whi5 is involved in coordinating adequate ribosome synthesis to G1 cell cycle progression. Whi5 is the yeast functional equivalent to the tumor suppressor Rb. This family of tumor suppressor genes is mutated in many types of cancers and is important in regulation of rRNA synthesis (Ruggero and Pandolfi, 2003). Several studies show a close relationship between Rb and transcription of the rDNA by RNA polymerase I (Cavanaugh et al., 1995; Hannan et al., 2000; Ciarmatori et al., 2001), and deleting Rb leads to increases in rRNA synthesis (Ciarmatori et al., 2001). Thus Whi5 and Rb have roles in both ribosome biogenesis and Start, although the regulatory connections may differ. In the future, it will be interesting to know if the analogy between Whi5 and Rb can be extended to the response to (and perhaps even regulation of) ribosome biogenesis.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
These authors contributed equally to this work.
Address correspondence to: Susan Baserga (susan.baserga{at}yale.edu)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bernstein, K. A. and Baserga, S. J. (2004). The small subunit processome is required for cell cycle progression at G1. Mol. Biol. Cell 15, 5038–5046.
Bernstein, K. A., Gallagher, J. E., Mitchell, B. M., Granneman, S., Baserga, S. J. (2004). The small-subunit processome is a ribosome assembly intermediate. Eukaryot. Cell 3, 1619–1626.
Burton, J. L. and Solomon, M. J. (2000). Hsl1p, a Swe1p inhibitor, is degraded via the anaphase-promoting complex. Mol. Biol. Cell 20, 4614–4625.
Cavanaugh, A. H., Hempel, W. M., Taylor, L. J., Rogalsky, V., Todorov, G., Rothblum, L. I. (1995). Activity of RNA polymerase I transcription factor UBF blocked by Rb gene product. Nature 374, 177–180.[CrossRef][Medline]
Ciarmatori, S., Scott, P. H., Sutcliffe, J. E., McLees, A., Alzuherri, H. M., Dannenberg, J. H., te Riele, H., Grummt, I., Voit, R., White, R. J. (2001). Overlapping functions of the pRb family in the regulation of rRNA synthesis. Mol. Cell. Biol 21, 5806–5814.
Costanzo, M., Nishikawa, J. L., Tang, X., Millman, J. S., Schub, O., Breitkreuz, K., Dewar, D., Rupes, I., Andrews, B., Tyers, M. (2004). CDK activity antagonizes Whi5, an inhibitor of G1/S transcription in yeast. Cell 117, 899–913.[CrossRef][Medline]
Cross, F. R. (1988). DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae. Mol. Cell. Biol 8, 4675–4684.
Cross, F. R. and Blake, C. M. (1993). The yeast Cln3 protein is an unstable activator of Cdc28. Mol. Cell. Biol 13, 3266–3271.
de Bruin, R. A., McDonald, W. H., Kalashnikova, T. I., Yates, J. 3rd, Wittenberg, C. (2004). Cln3 activates G1-specific transcription via phosphorylation of the SBF bound repressor Whi5. Cell 117, 887–898.[CrossRef][Medline]
Dolznig, H., Grebien, F., Sauer, T., Beug, H., Mullner, E. W. (2004). Evidence for a size-sensing mechanism in animal cells. Nat. Cell Biol 6, 899–905.[CrossRef][Medline]
Donachie, W. D. (1968). Relationship between cell size and time of initiation of DNA replication. Nature 219, 1077–1079.[CrossRef][Medline]
Dosil, M. and Bustelo, X. R. (2004). Functional characterization of Pwp2, a WD family protein essential for the assembly of the 90 S pre-ribosomal particle. J. Biol. Chem 279, 37385–37397.
Dragon, F., et al. (2002). A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. Nature 417, 967–970.[CrossRef][Medline]
Du, Y. C. and Stillman, B. (2002). Yph1p, an ORC-interacting protein: potential links between cell proliferation control, DNA replication, and ribosome biogenesis. Cell 109, 835–848.[CrossRef][Medline]
Dunbar, D. A., Wormsley, S., Agentis, T. M., Baserga, S. J. (1997). Mpp10p, a U3 small nucleolar ribonucleoprotein component required for pre-18S rRNA processing in yeast. Mol. Cell. Biol 17, 5803–5812.[Abstract]
Fingerman, I., Nagaraj, V., Norris, D., Vershon, A. K. (2003). Sfp1 plays a key role in yeast ribosome biogenesis. Eukaryotic Cell 2, 1061–1068.
Gallagher, J. E., Dunbar, D. A., Granneman, S., Mitchell, B. M., Osheim, Y., Beyer, A. L., Baserga, S. J. (2004). RNA polymerase I transcription and pre-rRNA processing are linked by specific SSU processome components. Genes Dev 18, 2506–2517.
Goldstein, A. L. and McCusker, J. H. (1999). Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541–1553.[CrossRef][Medline]
Grandi, P., et al. (2002). 90S pre-ribosome include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors. Mol. Cell 10, 105–115.[CrossRef][Medline]
Han, B. K., Aramayo, R., Polymenis, M. (2003). The G1 cyclin Cln3p controls vacuolar biogenesis in Saccharomyces cerevisiae. Genetics 165, 467–476.
Han, B. K., Bogomolnaya, L. M., Totten, J. M., Blank, H. M., Dangott, L. J., Polymenis, M. (2005). Bem1p, a scaffold signaling protein, mediates cyclin-dependent control of vacuolar homeostasis in Saccharomyces cerevisiae. Genes Dev 19, 2606–2618.
Hannan, K. M., Kennedy, B. K., Cavanaugh, A. H., Hannan, R. D., Hirschler-Laszkiewicz, I., Jefferson, L. S., Rothblum, L. I. (2000). RNA polymerase I transcription in confluent cells: Rb downregulates rDNA transcription during confluence-induced cell cycle arrest. Oncogene 19, 3487–3497.[CrossRef][Medline]
Hartwell, L. H., Culotti, J., Pringle, J. R., Reid, B. J. (1974). Genetic control of the cell division cycle in yeast. Science 183, 46–51.
Hartwell, L. H. and Unger, M. W. (1977). Unequal division in Saccharomyces cerevisiae and its implications for the control of cell division. J. Cell Biol 75, 422–435.
Johnston, G. C., Pringle, J. R., Hartwell, L. H. (1977). Coordination of growth with cell division in the yeast Saccharomyces cerevisiae. Exp. Cell Res 105, 79–98.[CrossRef][Medline]
Jorgensen, P., Nishikawa, J. L., Breitkreutz, B. J., Tyers, M. (2002). Systematic identification of pathways that couple cell growth and division in yeast. Science 297, 395–400.
Jorgensen, P., Rupes, I., Sharom, J. R., Schneper, L., Broach, J. R., Tyers, M. (2004a). A dynamic transcriptional network communicates growth potential to ribosome synthesis and critical cell size. Genes Dev 18, 2491–2505.
Jorgensen, P. and Tyers, M. (2004). How cells coordinate growth and division. Curr. Biol 14, R1014–1027.[CrossRef][Medline]
Jorgensen, P., Tyers, M., Warner, J. R. (2004b). Forging the factory: ribosome synthesis and growth control in budding yeast. In: Cell Growth, ed. M. N. Hall, M. Raff, G. Thomas. New York: Cold Spring Harbor Laboratory Press, 329–370.
Klionsky, D. J. (2005). Autophagy. Curr. Biol 15, R282–R283.[Medline]
Kushnirov, V. V. (2000). Rapid and reliable protein extraction from yeast. Yeast 16, 857–860.[CrossRef][Medline]
Lee, S. J. and Baserga, S. J. (1997). Functional separation of pre-rRNA processing steps revealed by truncation of the U3 small nucleolar ribonucleoprotein component, Mpp10. Proc. Natl. Acad. Sci. USA 94, 13536–13541.
Lee, S. J. and Baserga, S. J. (1999). Imp3p and Imp4p: two specific components of the U3 small nucleolar ribonucleoprotein that are essential for pre-18S rRNA processing. Mol. Cell. Biol 19, 5441–5452.
Longtine, M. S., McKenzie, A. R., Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961.[CrossRef][Medline]
Marion, R. M., Regev, A., Segal, E., Barash, Y., Koller, D., Friedman, N., O'Shea, E. K. (2004). Sfp1 is a stress- and nutrient-sensitive regulator of ribosomal protein gene expression. Proc. Natl. Acad. Sci. USA 101, 14315–14322.
Miller, M. E., Cross, F. R., Groeger, A. L., Jameson, K. L. (2005). Identification of novel and conserved functional and structural elements of the G1 cyclin Cln3 important for interactions with the CDK Cdc28 in Saccharomyces cerevisiae. Yeast 22, 1021–1036.[CrossRef][Medline]
Montagne, J., Stewart, M. J., Stocker, H., Hafen, E., Kozma, S. C., Thomas, G. (1999). Drosophila S6 kinase: a regulator of cell size. Science 285, 2126–2129.
Moore, S. A. (1988). Kinetic evidence for a critical rate of protein synthesis in the Saccharomyces cerevisiae yeast cell cycle. J. Biol. Chem 263, 9674–9681.
Nash, R., Tokiwa, G., Anand, S., Erickson, K., Futcher, A. B. (1988). The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog. EMBO J 7, 4335–4346.[Medline]
Nurse, P. and Thuriaux, P. (1977). Controls over the timing of DNA replication during the cell cycle of fission yeast. Exp. Cell Res 107, 365–375.[CrossRef][Medline]
Oeffinger, M. and Tollervey, D. (2003). Yeast Nop15p is an RNA-binding protein required for pre-rRNA processing and cytokinesis. EMBO J 22, 6573–6583.[CrossRef][Medline]
Oliver, E. R., Saunders, T. L., Tarle, S. A., Glaser, T. (2004). Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse Minute. Development 131, 3907–3920.
Pestov, D. G., Stockelman, M. G., Strezoska, Z., Lau, L. F. (2001a). ERB1, the yeast homolog of mammalian Bop1, is an essential gene required for maturation of the 25S and 5.8S ribosomal RNAs. Nucleic Acids Res 29, 3621–3630.
Pestov, D. G., Strezoska, Z., Lau, L. F. (2001b). Evidence of p53-dependent cross-talk between ribosome biogenesis and the cell cycle: effects of nucleolar protein Bop1 on G(1)/S transition. Mol. Cell. Biol 21, 4246–4255.
Polymenis, M. and Schmidt, E. V. (1997). Coupling of cell division to cell growth by translational control of the G1 cyclin CLN3 in yeast. Genes Dev 11, 2522–2531.
Popolo, L., Vanoni, M., Alberghina, L. (1982). Control of the yeast cell cycle by protein synthesis. Exp. Cell Res 142, 69–78.[CrossRef][Medline]
Ruggero, D. and Pandolfi, P. P. (2003). Does the ribosome translate cancer? Nat. Rev. Cancer 3, 179–192.
Ruvinsky, I., Sharon, N., Lerer, T., Cohen, H., Stolovich-Rain, M., Nir, T., Dor, Y., Zisman, P., Meyuhas, O. (2005). Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis. Genes Dev 19, 2199–2211.
Saracino, F., Bassler, J., Muzzini, D., Hurt, E., Carbone, M.L.A. (2004). The yeast kinase Swe1 is required for proper entry into cell cycle after arrest due to ribosome biogenesis and protein synthesis defects. Cell Cycle 3, 648–654.[Medline]
Schäfer, T., Straub, D., Petfalski, E., Tollervey, D., Hurt, E. (2003). The path from nucleolar 90S to cytoplasmic 40S pre-ribosomes. EMBO J 22, 1370–1380.[CrossRef][Medline]
Shima, H., Pende, M., Chen, Y., Fumagalli, S., Thomas, G., Kozma, S. C. (1998). Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase. EMBO J 17, 6649–6659.[CrossRef][Medline]
Strezoska, Z., Pestov, D. G., Lau, L. F. (2002). Functional inactivation of the mouse nucleolar protein Bop1 inhibits multiple steps in pre-rRNA processing and blocks cell cycle progression. J. Biol. Chem 277, 29617–29625.
Tyers, M., Tokiwa, G., Futcher, B. (1993). Comparison of the Saccharomyces cerevisiae G1 cyclins: Cln3 may be an upstream activator of Cln1, Cln2 and other cyclins. EMBO J 12, 1955–1968.[Medline]
Tyers, M., Tokiwa, G., Nash, R., Futcher, B. (1992). The Cln3-Cdc28 kinase complex of S. cerevisiae is regulated by proteolysis and phosphorylation. EMBO J 11, 1773–1784.[Medline]
Unger, M. W. and Hartwell, L. H. (1976). Control of cell division in Saccharomyces cerevisiae by methionyl-tRNA. Proc. Natl. Acad. Sci. USA 73, 1664–1668.
Wijnen, H. and Futcher, B. (1999). Genetic analysis of the shared role of CLN3 and BCK2 at the G(1)-S transition in Saccharomyces cerevisiae. Genetics 153, 1131–1143.
This article has been cited by other articles:
|
|
 |
|
  T. Pederson and R. Y.L. Tsai In search of nonribosomal nucleolar protein function and regulation J. Cell Biol., March 23, 2009; 184(6): 771 - 776. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Endo, M. Matsumoto, T. Inada, A. Yamamoto, K. I. Nakayama, N. Kitamura, and M. Komada Nucleolar structure and function are regulated by the deubiquitylating enzyme USP36 J. Cell Sci., March 1, 2009; 122(5): 678 - 686. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Shen, L. E. Cowen, A. M. Griffin, L. Chan, and J. R. Kohler The Candida albicans pescadillo homolog is required for normal hypha-to-yeast morphogenesis and yeast proliferation PNAS, December 30, 2008; 105(52): 20918 - 20923. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R. Houchens, A. Perreault, F. Bachand, and T. J. Kelly Schizosaccharomyces pombe Noc3 Is Essential for Ribosome Biogenesis and Cell Division but Not DNA Replication Eukaryot. Cell, September 1, 2008; 7(9): 1433 - 1440. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kundu-Michalik, M.-A. Bisotti, E. Lipsius, A. Bauche, A. Kruppa, T. Klokow, G. Kammler, and J. Kruppa Nucleolar Binding Sequences of the Ribosomal Protein S6e Family Reside in Evolutionary Highly Conserved Peptide Clusters Mol. Biol. Evol., March 1, 2008; 25(3): 580 - 590. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Yamada, C. Horigome, T. Okada, C. Shirai, and K. Mizuta Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity RNA, November 1, 2007; 13(11): 1977 - 1987. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. V. Rosado, D. Kressler, and J. d. l. Cruz Functional analysis of Saccharomyces cerevisiae ribosomal protein Rpl3p in ribosome synthesis Nucleic Acids Res., June 13, 2007; (2007) gkm388v1. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
