The small subunit processome is required for cell cycle progression at G1

Abstract

Without ribosome biogenesis, translation of mRNA into protein ceases and cellular growth stops. We asked whether ribosome biogenesis is cell cycle regulated in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and we determined that it is not regulated in the same manner as in metazoan cells. We therefore turned our attention to cellular sensors that relay cell size information via ribosome biogenesis. Our results indicate that the small subunit (SSU) processome, a complex consisting of 40 proteins and the U3 small nucleolar RNA necessary for ribosome biogenesis, is not mitotically regulated. Furthermore, Nan1/Utp17, an SSU processome protein, does not provide a link between ribosome biogenesis and cell growth. However, when individual SSU processome proteins are depleted, cells arrest in the G1 phase of the cell cycle. This arrest was further supported by the lack of staining for proteins expressed in post-G1. Similarly, synchronized cells depleted of SSU processome proteins did not enter G2. This suggests that when ribosomes are no longer made, the cells stall in the G1. Therefore, yeast cells must grow to a critical size, which is dependent upon having a sufficient number of ribosomes during the G1 phase of the cell cycle, before cell division can occur.

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 min with [³H]methyl-methionine, rRNA extracted, and analyzed by gel electrophoresis and blotting.

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. SSU processome proteins were tested for their ability to coimmunoprecipitate Mpp10 by Western blot with anti-Mpp10 antibody.

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 strains were used as controls. Net1-3xHA was immunoprecipitated with beads conjugated to anti-HA antibodies (IP, immunoprecipitation lane), and totals (T, total, representing 5% of the total protein extracted) were Western blotted with PAP antibodies, recognizing protein A. This blot was stripped and reprobed with anti-HA antibodies. Nan1-TAP was immunoprecipitated with IgG beads and Western blotted with anti-HA antibodies. Beads alone (BA) was used as a control.

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 into glucose media (depleted) for 24 h. YPH499 (the parent untagged strain) was used as a control. After 0, 3, 6, 12, and 24 h of depletion, cells were analyzed for growth by OD₆₀₀ (A) and for protein expression by Western blotting with anti-HA antibodies (B).

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 (depleted) for 24 h. YPH499 (the parent untagged strain) and Rps14A (a redundant ribosomal protein) were used as controls. Cells were stained with propidium iodide and FACS sorted.

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) for 23 h. YPH499 (the parent untagged strain) and GAL::3xHA-NET1 (a protein required for mitotic exit) were used as controls. Immunofluorescence was carried out using antibodies to tubulin and detected with TRITC (green), and Mpp10 was detected with FITC (red). DAPI (blue) was used to stain the cellular DNA.

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) and then shifted to glucose (depleted) for 21 h. a-Factor (2 μg/ml) was added to cells for 2.5 h and then washed out of the media. (A) Western blot on synchronized and released yeast. Protein was extracted from equal amounts of cells every fifteen minutes after the α-factor release and Western blotted with PAP antibodies (to visualize Cln2-TAP) and anti-HA antibodies (to visualize Clb2-3xHA). (B) FACS analysis of synchronized and released yeast. Cells were arrested with α-factor for 2.5 h and analyzed for DNA content (1C, 1 DNA content; 2C, 2 DNA content) by FACS. After 105 min of release from α-factor, yeast were again analyzed for DNA content by FACS sorting.