The ribosome biogenesis factor Rrp12 is required for S-phase progression. (A) Experimental plan used to examine S-phase progression in degron strains. (B) Cell cycle progression of degron strains after HU arrest at the restrictive temperature. DNA content of samples from the control and degron strains at different stages of the HU arrest experiment at 37°C (treatment 2 outlined in panel A). Asyn, asynchronously growing. (C) Percentages of cells with 1C DNA content at the different stages of the experiment described in the legend to panel B (top) and of the parallel experiment performed at 25°C (treatment 1 outlined in panel A) for two of the degron strains (bottom). (D) 25S/18S rRNA content of the control and degron strains when asynchronously growing (no HU) at 25°C and at the 45-min and 90-min time points of the experiment described in the legend to panel B. Total RNAs were prepared, and the levels of 25S rRNA relative to 18S rRNA quantitated on an Agilent bioanalyzer apparatus. (E) 20S/35S pre-rRNA content of control and rrp12-td strains when asynchronously growing (no HU) at 25°C and at the 45-min and 90-min time points of the experiment described in the legend to panel B. Total RNAs were analyzed by Northern blot analysis to detect the steady-state levels of 35S pre-rRNA, 20S pre-rRNA, and U1 spliceosomal snRNA. The 20S/35S ratios were quantified in an experiment that detected the 35S and 20S pre-rRNA contents with the same probe on the same membrane. Quantifications of 20S/U1 ratios were determined in an experiment that detected the contents of 20S pre-rRNA and U1 snRNA on the same membrane in two sequential hybridizations. The obtained values were plotted relative to the 20S/U1 ratio of control cells not treated with HU.

The Rrp12-td (DHFR^ts-Rrp12) protein expressed in the rrp12-td degron strain exhibits normal functionality for ribosome biogenesis. (A) Schematic diagrams of the wild-type Rrp12 protein and of the DHFR^ts-fused Rrp12 protein (Rrp12-td) expressed in the rrp12-td strain. The Rrp12-td protein carries an N-terminal extension of 209 amino acids that contains the sequence of a temperature-sensitive version of the mouse protein DHFR, a single copy of the c-MYC epitope, and a short flexible linker sequence with five repeats of the amino acid pair Gly-Ala. In addition, this Rrp12 protein has a deletion of the leucine residue at position 9, an artifact that was probably generated during the integration of the degron cassette into the RRP12 locus. This deletion by itself does not alter the functionality of the Rrp12 protein (see text for details). (B) The Rrp12-td protein present in the rrp12-td strain is expressed at lower levels than the endogenous Rrp12 present in control cells. (Left) Triton X-100 soluble lysates and TCA-precipitated whole-protein preparations from rrp12-GFP and rrp12-td–GFP cells were analyzed by Western blot analysis with anti-GFP and anti-Nop1 antibodies. (Right) Wild-type cells carrying a galactose-inducible RRP12-GFP plasmid (pGAL1-RRP12-GFP) or a galactose-inducible rrp12-td–GFP plasmid (pGAL1–rrp12-td–GFP) were grown to log phase in medium containing raffinose-glucose and then switched to medium containing galactose for 5 h. Total protein from cultures before and after galactose induction was precipitated by TCA and blotted with anti-GFP and anti-Nop1 antibodies. (C) The rrp12-td strain exhibits normal polysome content when cultured under permissive conditions. Cell extracts from control RRP12 cells cultured at 25°C and from rrp12-td cells after being cultured at 25°C or after being shifted from 25°C to 37°C for 2 h were resolved in sucrose linear gradients to determine the content of 40S, 60S, and 80S particles and polysomes. (D) The rrp12-td strain exhibits normal ratios and levels of rRNAs when maintained under permissive conditions. Total RNAs from the control and the indicated degron strains were prepared when cells were asynchronously growing (Asyn) at 25°C and after they were presynchronized with α-factor and released into HU-containing media (HU) for 2 h at 25°C. The levels of 25S rRNA relative to 18S rRNA and of rRNAs (25S + 18S) relative to total RNA were quantitated on an Agilent bioanalyzer apparatus. (E) Protein translation is not compromised in rrp12-td cells cultured under permissive conditions. Western blot analysis with anti-Mcm2, anti-Mcm7, anti-Rad53, anti-histone H3, anti-Nop1, and anti-Cdc11 of TCA-precipitated whole-protein preparations from control, utp5-td, and rrp12-td strains maintained at 25°C or shifted to 37°C for 1.5 h. (F) Sedimentation behavior of wild-type Rrp12 and Rrp12-td proteins. Lysates were prepared from cultures at 25°C of rrp12-MYC and rrp12-td strains carrying a KAP121-GFP plasmid and fractionated by ultracentrifugation on 7 to 50% sucrose gradients. After ultracentrifugation, fractions were collected, and the distribution of Rrp12 (first blot, top) and Rrp12-td (first blot, bottom) analyzed by anti-MYC immunoblotting. The same fractions were also probed with anti-Nop1 and anti-GFP antibodies to obtain the sedimentation profiles of Nop1 and Kap121, respectively. For each panel, the number of gradient fractions and the position of 40S particles are indicated at the bottom of the Kap121 blot. (G) The Rrp12-td protein is normally localized. Micrographs illustrating the subcellular localization of Rrp12-GFP and Rrp12-td–GFP proteins in wild-type cells carrying a galactose-inducible RRP12-GFP plasmid (pGAL1-RRP12-GFP) or a galactose-inducible rrp12-td–GFP plasmid (pGAL1–rrp12-td–GFP) after being grown to log phase in raffinose-glucose-containing media and then switched to galactose-containing media for 24 h at 25°C.

Rrp12 plays an active role in Kap121-mediated functions. (A) Nuclear import of Rrp12 in kap121^ts mutant cells. Control and Kap121^ts-expressing cells (pse1-1) carrying a GAL1-RRP12-GFP plasmid were grown in medium containing raffinose-glucose at 25°C and then were either transferred to medium containing galactose at 25°C for 3 h or, alternatively, shifted to 37°C for 2 h before being transferred to galactose-containing medium at 37°C for 3 h. Cells were photographed for Rrp12-GFP localization (left). The same experimental conditions were used for control and pse1-1 cells carrying either a GFP-histone H3 NLS-encoding plasmid (middle) or a GFP-Ulp1-encoding vector (right). (B) Overexpression of Rrp12 suppresses the temperature sensitivity of kap121^ts cells. pse1-1 cells were transformed with an empty GAL1 plasmid, a plasmid with the KAP121 gene, a plasmid with the KAP123 gene, or galactose-inducible expression plasmids for RRP12, rrp12-td, TSR1, or NOP1. Log-phase cultures of transformants were spotted at 10-fold serial dilutions onto galactose-containing plates, incubated at the indicated temperature (top) for 3 days, and photographed to assess cell growth. (C) Overexpression of Rrp12 restores proper localization of a histone H3 reporter, but not of Ulp1, in kap121 mutant cells. kap121^ts cells (pse1-1) carrying a GFP-histone H3 reporter plasmid (left panels) or a GFP-ULP1 plasmid (right panels) and also containing either an empty GAL1 vector or a GAL1-RRP12 plasmid were treated and analyzed by fluorescence microscopy at 25°C and 37°C as indicated in the legend to panel A. (D) Expression of Kap121 in rrp12-td cells. Triton X-100 soluble lysates (Triton) and TCA-precipitated whole-protein preparations (TCA) from control and rrp12-td cells carrying a KAP121-GFP plasmid were analyzed by Western blot analysis with anti-GFP (top) and anti-Nop1 (bottom) antibodies. (E) Kap121-GFP localization was analyzed with control and rrp12-td cells carrying a KAP121-GFP plasmid when asynchronously growing (left), after being arrested in G₁ by α-factor (middle), or after being arrested in early S phase using HU (right). Bars, 5 μm (A and C) and 2 μm (E).

Upregulation of dNTP synthesis is delayed in rrp12-td cells. (A) Experimental plan used to study dNTP production during the cell cycle. (B to H) Cells treated as described in panel A were collected at 20-min intervals for determination of dNTP content (B to F) and flow cytometry analysis (G) and at 10-min intervals for Western blot analysis with anti-Clb5 (H, top), anti-Sml1 (H, middle) and anti-Nop1 antibodies (H, bottom). Bar lines shown in panels B to F represent standard error values of the mean. (I) Quantification of relative protein contents in the experiment described in the legend to panel G. Protein levels are represented as fold change values relative to the point at which the protein content is the lowest.

Rrp12 is required for normal S-phase entry, mitosis, and activation of the DNA damage checkpoint. (A and B) Experimental plan used to examine the behavior of the rrp12-td and other degron strains during S-phase entry (A) and passage through mitosis (B). (C and D) Percentages of cells with 1C DNA content in the indicated strains at different stages of treatment 2 (C) and treatment 1 (D) outlined in panel A. (E) Percentage of cells with 1C DNA content in the indicated strains at different stages of treatment 2 outlined in panel B. (F) 20S/35S pre-rRNA content in control, utp18-td, rrp12-td, and utp5-td cells when α-factor arrested (time zero) and at the 45-min time point shown in panel C. Total RNAs were analyzed by Northern blot analysis to detect the steady-state levels of 35S pre-rRNA, 20S pre-rRNA, and U1 spliceosomal snRNA. Quantification of 20S/35S and 20S/U1S ratios was performed as described in the legend to Fig. 1E. (G) Sensitivity of rrp12-td cells to DNA damage. Cultures of the indicated yeast strains were spotted at 10-fold serial dilutions onto YPD plates not supplemented (No Cu²⁺) or supplemented with CuSO₄ (Cu²⁺) that contained no drugs, HU, or MMS at the indicated concentrations. Plates were scored for growth after incubation at the indicated temperatures for 3 days. (H) Outline of the experimental plan to study the activation of the DNA checkpoint. (I) Western blot analysis with the indicated antibodies of TCA-precipitated whole-protein preparations from the indicated strains treated as outlined in the legend to panel G. (J) Western blot analysis with the indicated antibodies of TCA-precipitated whole-protein preparations from control and rrp12-td strains maintained at 25°C or after a shift to 37°C for 1.5 h.

Rrp12 interacts with Kap121. Proteins associated with Rrp12-GFP or Rrp12-td-GFP (DHFR^ts-Rrp12-GFP) purified with GFP-Trap from unfractionated lysates (A) or lysates that had been depleted of high-molecular-weight complexes by ultracentrifugation (B) were identified by mass spectrometry as indicated in Materials and Methods. Proteins associated with Kap121-GFP (C) and Kap123-GFP (D) were purified from RRP12 and rrp12-td cells carrying either a KAP121-GFP-carrying or a KAP123-GFP-carrying plasmid, respectively, and identified by mass spectrometry. Triangles indicate nonspecific proteins commonly found in purifications using the GFP-Trap technique.

Rrp12 and Kap121 are required for nuclear localization of the Rnr2/Rnr4 complex. (A, B) Nuclear import of Rnr4 in rrp12-td cells. Control and rrp12-td cells containing either a GAL1-GFP-RNR4 plasmid or a GAL1-TSR1-GFP plasmid were transferred from medium containing raffinose-glucose to medium containing galactose at 25°C for 5 h before being photographed to detect the localization of GFP-tagged proteins. Photographs were obtained in a conventional fluorescence microscope (A) and a confocal microscope (B). Graphs show the quantification analysis of GFP fluorescence across 4-μm sections in four individual cells which are indicated by red and blue lines on the photographs. (C) Galactose-induced expression of GFP-Rnr4 in rrp12-td cells. Cells at the indicated time points after galactose induction were analyzed by Western blot analysis with anti-GFP (top) and anti-Nop1 (bottom) antibodies. (D) Western blot analysis of rnr2-MYC, rnr2-MYC/rrp12-td, rnr4-MYC, and rnr4-MYC/rrp12-td strains with anti-MYC antibodies (top). We examined the expression levels of Nop1, as a loading control, in each sample (bottom). (E, F) Quantification of Rnr2-MYC and Rnr4-MYC localization in rnr2-MYC, rnr2-MYC/rrp12-td, rnr4-MYC, and rnr4-MYC/rrp12-td strains. Protein localization was visualized on cells exponentially growing at 25°C by anti-MYC indirect immunofluorescence. Cells were stained with DAPI to examine the localization of the MYC-tagged proteins relative to the nucleus. A minimum of 200 cells were counted in three independent experiments to calculate average values. Bars shown in panel F represent standard error values. (G) Interaction of Rrp12 and Rnr2. rnr2-MYC and rnr4-MYC cells carrying either an empty GAL1 vector, a GAL1-RRP12-GFP plasmid, or a GAL1-rrp12-td-GFP plasmid were transferred from medium containing raffinose-glucose to medium containing galactose for 3 h at 25°C. Total cellular extracts (TCL) and GFP-Trap-purified samples (GFP-Trap) were prepared and analyzed by Western blot analysis with anti-GFP (top) and anti-MYC (bottom) antibodies. Samples corresponding to rnr2-MYC cells are shown in lanes 1, 3, 5, 7, 9, and 12. The rest of the lanes correspond to samples of rnr4-MYC cells. (H) dNTP levels in control and rrp12-td cells when growing asynchronously (Asyn) and after being arrested in G₁ by α-factor at 25°C (α-factor). The relative difference in dNTP concentrations between control and rrp12-td strains, cultured under the same conditions, is shown on top of black bars. Bar lines indicate the standard error. *, P value of ≤0.05 compared to control sample; **, P value of ≤0.01 compared to the control sample; n.s., not statistically significant. The nonstatistically significant values obtained in the dGTP quantifications in α-factor-arrested cells are probably due to the low sensitivity of the technique for accurately detecting differences at very low concentration ranges. (I) Nuclear import of Rnr4 in Kap121^ts-expressing (pse1-1) cells. Indicated cells carrying either a GAL1-GFP-RNR4- or a GAL1-TSR1-GFP-carrying plasmid were grown in raffinose-glucose-containing medium at 25°C and then transferred to galactose-containing medium at 25°C for 3 h or, alternatively, shifted to 37°C for 2 h before being transferred to galactose-containing medium at 37°C for 3 h. The localization of GFP-tagged proteins was visualized by fluorescence microscopy. (J) Rrp12 promotes nuclear import of Rnr4 in kap121^ts cells. Kap121^ts-expressing cells (pse1-1) cotransformed with a GAL1-GFP-RNR4 plasmid and either a GAL1 empty vector or a GAL1-RRP12 plasmid were analyzed by fluorescence microscopy after inducing the expression of the proteins with galactose for 5 h at 25°C. Bars 2 μm (A, I, and J) and 2 μm (B).

Deletion of SML1 suppresses the defective response to DNA damage of rrp12-td cells. (A) Deletion of SML1 suppresses the DNA damage sensitivity of rrp12-td cells. Cultures of control, sml1Δ, rrp12-td, and sml1Δ rrp12-td strains were spotted at 10-fold serial dilutions onto YPD plates containing no drugs, HU, or MMS at the indicated concentrations, incubated at 25°C for 3 days, and photographed to assess cell growth. (B) Deletion of SML1 restores the activation of the DNA damage checkpoint in rrp12-td cells. Western blot analysis with anti-Rad53 (top), anti-Sml1 (middle), and anti-Mcm2 (bottom) antibodies of TCA-precipitated whole-protein preparations from rrp12-td, sml1Δ rrp12-td, and control strains arrested in G₁ with α-factor and released into HU-containing medium for the indicated periods of time.