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. 2017 Feb 21;8(8):13240-13252.
doi: 10.18632/oncotarget.14658.

The roles of RRP15 in nucleolar formation, ribosome biogenesis and checkpoint control in human cells

Zhixiong Dong  1   2 , Changjun Zhu  1   2 , Qimin Zhan  3 , Wei Jiang  1   3
Affiliations

The roles of RRP15 in nucleolar formation, ribosome biogenesis and checkpoint control in human cells

Zhixiong Dong et al. Oncotarget. .

Abstract

The nucleolus controls ribosome biogenesis and its perturbation induces nucleolar stress that inhibits cell cycle progression and activates checkpoint responses. Here, we investigate the roles of ribosomal RNA processing protein, RRP15, in nucleolar formation, ribosome biogenesis, cell cycle progression and checkpoint control in human cells. RRP15 is localized in the nucleolus and required for nucleolar formation. In contrast to the budding yeast Rrp15p that was reported as a component of pre-60S subunits, RRP15 is found in both pre-40S and pre-60S subunits and involved in regulating rRNA transcription and ribosome biogenesis. Perturbation of RRP15 induces nucleolar stress that activates RPL5/RPL11/5S rRNA (RP)-Mdm2-p53 axis checkpoint response and arrests cells at G1-G1/S in p53-proficient non-transformed RPE1 cells but not in p53-deficient HeLa and MCF7 tumor cells. Instead, p53-deficient HeLa and MCF7 cells with RRP15-dependent nucleolar stress enter S-phase with S-phase perturbation that activates ATR-Chk1- γH2AX axis DNA replication/damage checkpoint response, delaying S-G2/M progression and, ultimately, causing cell death. The selective checkpoint response, cell cycle inhibition and/or cytotoxicity induced by RRP15-dependent nucleolar stress in p53-proficient non-transformed cells and p53-deficient tumor cells suggest that RRP15 might be a potential target for cancer therapy.

Keywords: RRP15; checkpoint control; nucleolar stress; nucleolus; ribosome biogenesis.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Subcellular localization of RRP15
A. The schematic diagrams of RRP15 wild type (WT) and different mutants. B. Immunofluorescence analysis of HeLa cells with rabbit α-RRP15 and mouse α-nucleolin, α-fibrillarin or α-UBF. DNA was visualized by DAPI staining. R values were obtained as described (see supplemental Materials and Methods). Scale bars, 5 μm. C. HeLa cells were transfected with plasmids of pEGFP-RRP15WT, pEGFP-RRP15N150, pEGFP-RRP15C175, pEGFP-RRP15M43 or pEGFP- RRP15Δ228-232 for 48 h. Cell lysates were immunoblotted with mouse α-GFP. D. HeLa cells grown on coverslips were transfected with plasmids of pEGFP-RRP15WT, pEGFP-RRP15N150, pEGFP-RRP15C175, pEGFP-RRP15M43 or pEGFP- RRP15Δ228-232 for 48 h. The cells were fixed and stained with mouse α-nucleolin and DAPI. Scale bars, 5 μm. R values were obtained as described in (B).
Figure 2
Figure 2. Nucleolar formation and nucleolin, fibrillarin and UBF localization in cells depleted RRP15
A. HeLa cells were transfected with mock (buffer), control siRNA, RRP15 siRNA or RRP15 esiRNA for 48 h and cell lysates were immunoblotted with α-RRP15 and anti-α-Tubulin antibody. B-D. HeLa cells grown on coverslips were transfected with or without RRP15 siRNA for 48 h, fixed and immunostained with α-RRP15 and α-nucleolin, α-fibrillarin or α-UBF. DNA was visualized by DAPI staining. Scale bars, 5 μm. Intensities of nucleolin, fibrillarin or UBF staining in the nucleoli and the nucleoplasm in 20 cells were determined by Image-Pro Plus 7.0. ** P<0.01.
Figure 3
Figure 3. Presentation and regulation of RRP15 in pre-40S and pre-60S ribosomal subunits and ribosome biogenesis
A. Pre-ribosomal profile in HeLa cells. Nuclear extracts were prepared from HeLa cells and fractionized on 10% to 40% sucrose density gradient. The absorbance at 260 nm (A260) of each fraction was profiled and the positions of pre-ribosomal subunits were indicated. B. RRP15 in pre-ribosomal subunits. Proteins from fractions described in (A) were separated on a SDS-PAGE and immunoblotted with α-RRP15, α-eIF6 (pre-60S ribosomal subunit protein) and α-Nucleolin (nucleolar protein). C. Ribosome biogenesis impaired by RRP15 depletion. HeLa cells transfected with or without RRP15 siRNA for 48 h or HeLa cells were treated with ActD for 12 h (positive control) were collected. Pre-ribosomal subunits were purified from these cells and profiled as described in (A). RRP15 depletion was determined by immunoblotting analysis (insert).
Figure 4
Figure 4. Regulation of 47S pre-rRNA transcription by RRP15
A. The effects of RRP15 on 47S pre-rRNA transcription. Total RNA from HeLa cells transfected with or without RRP15 siRNA for 48 h or treated with ActD for 12 h (positive control) was extracted and 47S pre-rRNA was amplified by real-time RT-PCR using specific primers as indicated in the diagram of 47S pre-rRNA. Histograms represented 47S pre-rRNA levels in HeLa cells treated with control, RRP15 siRNA and ActD. B. Determination of RRP15 binding sites in rDNA loci by ChIP analysis. HeLa cells were cross-linked and immunoprecipitated by IgG (negative control), α-UBF (positive control) or α-RRP15. DNA in IgG, α-UBF or α-RRP15 immunoprecipitates was amplified by indicated PCR primers and then separated by agrose gel electrophoresis. Histograms represented the percentages of DNA immunoprecipitated by α-RRP15 and α-UBF that were relative to input DNA. C. A schematic representation of human rDNA repeats with RRP15 binding sites. Green circle dots represented RRP15 binding sites and red circle dots represented RRP15 negative binding sites. Primer pairs (black solid bars) and their approximate positions relative to the transcription start are indicated. D. Perturbation of UBF and RPA194 association with rDNA loci by RRP15 depletion. HeLa cells transfected with or without RRP15 siRNA for 48 h were employed to perform ChIP analysis described in (B) using α-UBF and α-RPA194. Histograms represented the percentages of DNA immunoprecipitated by α-UBF and α-RPA194 that were relative to input DNA in cells transfected with or without RRP15 siRNA. *P<0.05 and **P<0.01.
Figure 5
Figure 5. The effects of RRP15 ablation on cell proliferation and cell cycle progression in various human cells
A. Inhibition of cell proliferation by RRP15 depletion in various human cells. RPE1 (up), HeLa (middle) and MCF7 cells (low) were transfected with or without RRP15 siRNA and cell proliferation was determined by MTT assay at indicated times. Results represent means ± standard deviations of five independent experiments. B. RPE1 (up), HeLa (middle) and MCF7 cells (low) were transfected with or without RRP15 siRNA for 48 h and cell cycle profiles were monitored by flow cytometry. Results are representative of three independent experiments. C. RPE1, HeLa and MCF7 cells were transfected with or without RRP15 siRNA for 48 h. The cell lysates were immunoblotted with α-RRP15, α-Caspase 3 and anti-α-Tubulin antibody. D. The effects of RRP15 depletion on BrdU (5-bromo-2′-deoxyuridine) incorporation in various human cells. Cells transfected with or without RRP15 siRNA for 48 h were labeled with 10 mM BrdU (1 h). The cells were fixed and immunostained with mouse α-BrdU. DNA was visualized by DAPI staining.
Figure 6
Figure 6. Induction of nucleolar stress response in p53-proficient RPE1 cells or S-phase checkpoint response in p53-deficient HeLa or MCF7 cells by RRP15 ablation
A. RRP15 depletion in RPE1 cells enhanced Mdm2 interaction with RPL11. RPE1 cells were transfected with or without RRP15 siRNA for 48 h. Whole-cell lysates were subjected to immunoprecipitation and immunoblotting analyses. B and C. The effects of RRP15 depletion on expression of cell cycle related proteins in various human cells. RPE1, HeLa or MCF7 cells were transfected with or without RRP15 siRNA for 48 h. Whole cell lysates were immunoblotted with indicated antibodies. D. The effects of RRP15 depletion on the expression of DNA damage related proteins in various human cells. HeLa or MCF7 cells were transfected with or without RRP15 siRNA for 48 h. Whole cell lysates were immunoblotted with indicated antibodies. E and F. RRP15 depletion induced increases of γH2AX level and γH2AX foci formation in HeLa and MCF7 cells. RPE1, HeLa or MCF7 cells transfected with or without RRP15 siRNA for 48 h were immunoblotted or immunostained with indicated antibodies. Scale bars, 5 μm. G. Histograms represented average numbers of γH2AX foci detected in 20 cells in (F). ***P<0.001. H. The model of RRP15 involved in regulating ribosome biogenesis and cell proliferation (for detail, see text).
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