Because it is well known that ribosome biogenesis ceases during mitosis in mammalian cells, we sought to determine whether rRNA is made and processed during mitosis in yeasts. To do this, we pulsed interphase (untreated cells) or mitotically arrested yeast cells with [3H]methyl-methionine and looked for defects in transcription and processing of pre-rRNA (). Cell cycle arrest was verified by light microscopy that showed that >90% of the cells were arrested as large-budded cells, indicating a block at G2/M (our unpublished data). Equal amounts of tritiated RNA were resolved on denaturing agarose gels. These results show that mitotically arrested S. cerevisiae cells transcribe and process the nascent 35S pre-rRNA equally well during interphase and mitosis (). We also conducted similar experiments in S. pombe, because they are more similar to mammalian cells with respect to their cell cycle. As described above, equal amounts of tritiated RNA were resolved on denaturing agarose gels. We found that they, too, continue to make and process pre-RNA during mitosis (). Cell cycle arrest was again verified by light microscopy where 90% of the cells were arrested as large, elongated cells, indicating a block at G2/M (our unpublished data). Together, these results suggest that ribosome biogenesis continues during mitosis in the yeasts S. cerevisiae and S. pombe, unlike in metazoan cells.
Figure 1. rRNA transcription and processing occurs during mitosis in S. cerevisiae and S. pombe. Cells were grown in media lacking methionine for 48 h. Cells were arrested in mitosis with the drug nocodazole. Both interphase and mitotic cells were pulsed for 10 (more ...)
Next, we determined whether synthesis of the SSU processome is itself mitotically regulated in S. cerevisiae by testing whether it was intact during mitosis. This was done by assessing the ability of SSU processome proteins to associate with each other. To assess whether the SSU processome was intact, coimmunoprecipitation of 3xHA-tagged SSU processome proteins with Mpp10 was tested in interphase and mitotically arrested cells. Utp1, Utp2, Utp3, Utp4, Utp5, Utp6, Utp7, Utp8, Utp9, Utp10, Utp12, Utp13, Utp14, Utp15, Utp16, and Utp17 were all able to coimmunoprecipitate Mpp10 during interphase as well as during mitosis (). These results suggest that the SSU processome remains intact during mitosis.
Figure 2. The SSU processome remains intact in mitotic cells. 3xHA-tagged SSU processome proteins Utp1-10, 12-17 were immmunoprecipitated from glass bead extracts made from interphase or mitotically arrested cells, by using beads conjugated with anti-HA antibodies. (more ...)
Previous results indicated that one SSU processome component, Utp17/Nan1, might link ribosome biogenesis with the exit from mitosis (Shou et al., 1999
). Utp17 was originally named Nan1 for N
ucleolar protein 1. Utp17/Nan1 was originally purified as a Net1-associated protein through affinity chromatography (Shou et al., 1999
). Net1 is a component of the regulator of nucleolar silencing and telophase (RENT) complex, which is important for mitotic exit through its association with Cdc14 and silencing through its association with Sir2 (Shou et al., 1999
). Utp17/Nan1 was subsequently purified as a component of the SSU processome, the results were extensively validated, and it was shown to be required for generation of the 18S rRNA (Dragon et al., 2002
). YPH499, the untagged parent strain, is used as a control for this and subsequent experiments. We asked whether Utp17/Nan1 was also a true component of the RENT complex by examining whether it could coimmunoprecipitate Net1. We constructed double-tagged strains where Nan1 was tagged with the TAP tag (Rigaut et al., 1999
) and Net1 with 3xHA. When Net1–3xHA was immunoprecipitated and then Western blotted for Nan1, the same amount of Nan1 coimmunoprecipitated in the double-tagged strain as in the single Nan1-TAP strain (), thus representing background. We have confirmed that Net1–3xHA was enriched by coimmunoprecipitation by blotting with anti-HA antibodies (). We obtained the same result by coimmunoprecipitating Nan1-TAP first and then Western blotting for Net1–3xHA (). This suggests that Nan1 and Net1 do not coimmunoprecipitate. These results suggest that Nan1 is likely not Net1-associated during interphase and is therefore not a component of the RENT complex, contrary to what has been previously reported.
Figure 3. Net1 and Nan1 do not coimmunoprecipitate. Nan1/Net1 coimmunoprecipitation experiments were carried out with Net1-3xHA-, Nan1-TAP–, YPH499-, and Net1-3xHA/Nan1-TAP–tagged yeast strains. YPH499 (untagged parent strain), Net1-3xHA, and Nan1-TAP (more ...)
We determined whether ribosome biogenesis was required for cell cycle progression by asking whether depletion of individual SSU processome proteins would lead to arrest at a particular stage of the cell cycle. Because all but one of the SSU processome proteins are essential, their depletion ultimately affects cell growth. In these strains, the SSU processome proteins are under the control of a galactose-inducible promoter and tagged with a 3xHA tag. In the presence of galactose, the protein is made; however, in glucose, protein levels are depleted. Growth of cells depleted of SSU processome proteins begins to decline after 12 h of depletion (). Growth of cells depleted of Utp16, a nonessential SSU processome component, was only slightly slowed in comparison with the parent strain YPH499, as expected. After 24 h of protein depletion, the SSU processome components were not detectable by Western blot ().
Figure 4. Depletion of the essential SSU processome proteins slows growth. Strains expressing Utp1-17 from a galactose-inducible/glucose-repressible promoter with a 3xHA tag were grown to early log phase in galactose/raffinose media (undepleted) and then shifted (more ...)
The cell cycle profiles of SSU processome-depleted strains were compared with those obtained from control strains YPH499 (parent strain) and GAL::3xHA-RPS14A
, after growth in galactose and glucose. Rps14A is a redundant ribosomal protein; that is, depletion of one of the Rps14 proteins (Rps14A or Rps14B) does not appreciably affect cell growth. When the normally expressed Rps14A is depleted, protein levels of Rps14B are increased 10-fold (Paulovich et al., 1993
). Up-regulation of Rps14B protein levels occur through posttranslational modification of RPS14B
(Li et al., 1995
; Fewell and Woolford, 1999
). The growth of YPH499 and GAL::3xHA-RPS14A
in galactose yields a cell cycle profile indicative of cells primarily in the G1 phase of the cell cycle upon FACS sorting (, Undepleted). This is because galactose is a poor carbon source and the cells grow slowly. However, when these strains are shifted to glucose and analyzed by FACS sorting, the cell cycle profile for these two strains shifts to nearly equal G1 and G2 peaks (, Depleted). Thus, this is the expected cell cycle distribution result after the switch from galactose to glucose if cells are cycling normally. When SSU processome proteins are not depleted (Utp1-17 grown in galactose) the yeast strains yield cell cycle profiles identical to those obtained by FACS sorting of YPH499 and GAL::3xHA-RPS14A
, with a prominent G1 peak (, Undepleted). Cells with integrated galactose promoters grew slower in galactose media and had a more prominent G1 peak than YPH499. However, when these cells are shifted into glucose to deplete the indicated proteins and analyzed by FACS sorting, the cell cycle profiles are different from those obtained from cells that are cycling normally. In yeast depleted of SSU processome proteins Utp1-15, and Utp17 by growth in glucose (, Depleted) the G1 peak continues to be much larger than the G2 peak, with little change upon shift to glucose (compare Depleted YPH499 and Rps14A to the Utp proteins). This indicates arrest in the G1 phase of the cell cycle. In contrast, yeast depleted of Utp16, a nonessential SSU processome protein, yielded a cell cycle profile similar to the YPH499 and GAL::3xHA-RPS14A
strains, with equal G1 and G2 peaks (). Thus, as expected, depletion of the nonessential Utp16 protein does not cause arrest at G1.
Figure 5. Depletion of SSU processome proteins leads to G1 arrest. Strains expressing Utp1-17 and Rps14A from galactose-inducible/glucose-repressible promoters were grown in early log phase in galactose/raffinose media (undepleted) and then shifted into glucose (more ...)
The G1 arrest upon essential Utp protein depletion was further supported by lack of immunofluorescence of a marker for the G2 phase of the cell cycle (). Tubulin staining is indicative of progression to G2 phase of the cell cycle, because tubulin is only detectable in G2 when the mitotic spindle is present. YPH499, GAL::3xHA-NET1
, and GAL::3xHA-UTP18
were grown to early log phase in galactose-containing medium and then shifted to glucose-containing medium for 24 h. Cells were analyzed by immunoflouresence for the presence of cellular DNA (blue); Mpp10, an SSU processome component (red); and tubulin (green). As expected, both Mpp10 and tubulin were expressed in the parent YPH499 strains (). Similarly, cells depleted of Net1, a protein required for cell cycle progression at G2/M (Straight et al., 1999
) resulted in expression of Mpp10 and tubulin (). However, when Utp18 was depleted, Mpp10 and tubulin expression were no longer observed (). This suggests that these cells no longer form mitotic spindles and is consistent with an arrest in G1. Because Mpp10 is a component of the SSU processome, the lack of Mpp10 staining in Utp18-depleted cells suggests that the SSU processome is no longer intact. Therefore, the lack of ribosomes after depletion of SSU processome proteins leads to stalling in the G1 phase of the cell cycle. Yeast depleted of SSU processome proteins are able to form small buds, suggesting that these yeast may be stalled in late G1 (our unpublished data). Therefore, this suggests that the SSU processome is required for cell cycle progression at G1.
Figure 6. Immunofluorescence of depleted SSU processome proteins is consistent with G1 arrest. YPH499, GAL::3xHA-NET1, and GAL::3xHA-UTP18 yeast strains were grown to early log phase in galactose/raffinose media (undepleted) and then shifted into glucose (depleted) (more ...)
To further verify this arrest, we created yeast strains that could be synchronized and monitored for the expression of additional cell cycle markers. Yeast strains were created by the disruption of BAR1 that could be easily synchronized in G1 by the addition of low concentrations of the mating-type pheromone α-factor. In the Δbar1 strain, the cell cycle cyclins Cln2 and Clb2 were chromosomally tagged with TAP and 3xHA, respectively. Cln2 is a cell cyclin that is expressed during G1, whereas Clb2 is a cyclin that is expressed at G2. In normally cycling cells, after they are released from the G1 arrest, the cells will first express the cell cyclin Cln2-TAP and then express Clb2-3xHA as the cells progress into G2. The cells remain synchronized for one cell cycle (~100 min) after being released from α-factor. The necessary strains were constructed for GAL::UTP1, GAL::UTP2, GAL::UTP4, and GAL::RPS14A by deletion of Bar1 (Δbar1) and the integration of TAP tag and the HA tag at CLN2 (Cln2-TAP) and CLB2 (Clb2–3xHA) in each strain.
SSU processome-depleted cells were synchronized with α-factor and then released to determined whether cells depleted of SSU processome proteins were then able to enter G2. The strains (construction described in the above paragraph) were grown in medium containing galactose (Undepleted) until OD600 0.2–0.6. The cells were shifted into glucose media (Depleted) for 21 h and then arrested in G1 with α-factor for 2.5 h. Cells were released from the G1 arrest (by washing α-factor out of the media), and the extracts were analyzed by Western blot or the cells sorted by FACS. The same number of cells were collected every 15 min after the G1 arrest, protein extracted, and Western blotted for the G1 cyclin Cln2-TAP or the G2 cyclin Clb2-3xHA via their respective tags (). After release from the G1 arrest, cells depleted of Rps14A, a redundant nonessential ribosomal protein, expressed Cln2 beginning at ~30 min after release, and the levels of this cyclin declined over time. In this same strain, protein levels of Clb2 were visualized at ~75 min after release and remained constant after this time point. Expression of this protein demonstrated that these cells were cycling from G1 into the G2 stage of the cell cycle. In contrast, cells depleted of Utp1, Utp2, and Utp4 only expressed the G1 cyclin Cln2, although expression of this protein was variable from strain to strain (). Expression of the G2 cyclin could not be detected at all when these SSU processome components were depleted.
Figure 7. Synchronized depleted SSU processome proteins arrest in G1 upon cell cycle release. GAL::RPS14A, GAL::UTP1, GAL::UTP2, GAL::UTP4 yeast strains bearing Δbar1, Cln2-TAP, Clb2-3xHA were grown to early log phase in galactose/raffinose media (undepleted) (more ...)
The cell cycle arrest resulting from depletion of SSU processome components was further analyzed by FACS analysis of synchronized cells (). Cells arrested with α-factor for 2.5 h or released from this arrest for 105 min were collected and analyzed for one or two DNA contents (1C or 2C, respectively). After incubation with α-factor, Rps14A-, Utp1-, Utp2-, and Utp4-depleted cells had predominantly arrested in G1 (1C DNA at 0 min). When released from this arrest for 105 min, Rps14A-depleted cells were predominantly in G2 with a 2C DNA content at 105 min, whereas Utp1-, Utp2-, and Utp4-depleted cells were still arrested in G1 (1C at 105 min). Together, these results suggest that the SSU processome is required for cell cycle progression at G1.