![[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}
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Previous Article | Next Article 
Molecular and Cellular Biology, May 2007, p. 3682-3694, Vol. 27, No. 10
0270-7306/07/$08.00+0 doi:10.1128/MCB.00172-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Institute of Clinical Molecular Biology and Tumor Genetics,1 Institute of Molecular Immunology, GSF Research Center, Marchioninistrasse 25, D81377 Munich, Germany2
Received 30 January 2007/ Returned for modification 26 February 2007/ Accepted 1 March 2007
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We have recently characterized a nucleolar complex of endogenous Pes1, Bop1, and WDR12 in mammalian cells, termed the PeBoW complex (14). Interestingly, expression of N-terminal or C-terminal truncations of Pes1, Bop1, or WDR12 in mammalian cells blocked processing of the 32S pre-rRNA into mature 28S rRNA and triggered p53-dependent cell cycle arrest (10, 14, 20, 23). Apparently, the PeBoW complex is a good target for the generation of dominant-negative mutants and plays a crucial role in rRNA processing and maturation of the large ribosomal subunit.
Coimmunoprecipitation assays showed that dominant-negative mutant forms of Pes1 were indeed incorporated into the PeBoW complex, suggesting that they block its function by building up dead-end complexes that prevent further essential interactions (10, 20). Thus, the amount of PeBoW components needs to be tightly controlled and adjusted to the rate of ribosome synthesis and proliferation. Quiescent or serum-starved cells exhibit low levels of Pes1, Bop1, and WDR12 that are induced by the proto-oncogene c-Myc upon cell cycle entry (14). c-Myc is overexpressed in a variety of human malignancies, suggesting a coordinated upregulation of the PeBoW complex in tumor cells. But interestingly, amplification of the gene for Bop1, but not that for Pes1, was frequently found in colorectal cancers, associated with an increase in Bop1 mRNA (18). The PeBoW complex may play an additional role in mitosis, as transient overexpression of Bop1 increased the percentage of multipolar spindles. Depletion of Pes1 or Bop1 also caused an increase in abnormal mitotic figures (17).
These observations underline the importance of a functional PeBoW complex playing a role in the cross talk between ribosome biogenesis and the cell division cycle. However, it is unknown how the integrity of the PeBoW complex is controlled in mammalian cells.
In this study, we investigated how changes in the abundance of individual PeBoW components affect its functionality. We show that overexpression of Bop1 disturbs cell proliferation and ribosome biogenesis by titrating endogenous WDR12 into a Bop1/WDR12 subcomplex and that its negative effects could be rescued by coexpression of WDR12, but not Pes1. Further, Bop1 was found to be essential for the copurification of Pes1 and WDR12, thus arguing for an indirect interaction mediated by Bop1. Finally, depletion of individual PeBoW components by RNA interference revealed a strong interdependence of their protein levels. Thus, the integrity of the PeBoW complex is tightly controlled by protein-protein interactions and highly sensitive to elevated levels of Bop1.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
RNA analysis and 32P in vivo labeling. Total RNA was isolated with Trifast (PeqLab). Two micrograms of total RNA for detection of ITS-1 and ITS-2 or 10 µg of total RNA for analyzing the endogenous mRNA levels was separated on a 1% agarose-formaldehyde gel and blotted onto Hybond N+ membranes (GE Healthcare). The following 32P-end-labeled DNA oligonucleotides were used to visualize rRNA precursors: ITS-1 (human specific), 5'-CCTCCGCGCCGGAACGCGCTAGGTACCTGGACGGCGGGGGGGCGGACG-3'; ITS-2 (human specific), 5'-GCGGCGGCAAGAGGAGGGCGGACGCCGCCGGGTCTGCGCTTAGGGGGA-3'; ITS-1 (rat specific), 5'-GGACCAGACCCGACACCCTGCCACCGCACACCTGTCCCGAAACCCCCT-3'; ITS-2 (rat specific), 5'-GCCCCGGGGAGCGGGCCCTGCGAGCAGACTCCCAGCCGCGCGACGCGA-3'; 18S rRNA (human and rat specific), 5'-CACCCGTGGTCACCATGGTAGGCACGGCGACTACCATCGAAAGTTGATAG-3'.
Metabolic labeling of rRNA has been described elsewhere (14).
Production of antibodies. Monoclonal antibodies (MAbs) against human nucleostemin and human Nog1 were generated as previously described (14). For immunization, we used a glutathione S-transferase (GST)-nucleostemin fusion protein and a Nog1-specific peptide coupled to ovalbumin (Peptide Specialty Laboratories GmbH, Heidelberg, Germany). The Nog1 peptide sequence is ESKEKNTQGPRMPRTAKKVQRTVLEKC. The nucleostemin (7H3) MAb belongs to the immunoglobulin G2b subclass, and the Nog1 (1D8) MAb belongs to the immunoglobulin G2a subclass. Polyclonal antibodies against mouse Bop1 were raised by immunization of guinea pigs with a mixture of the peptides GKPHMSPASLPGKRRLEPDQELQIQ and SQEHTQVLLHQVSRRRSQSPFRRSHG.
Immunoblotting, immunofluorescence, and immunoprecipitation. For immunoblotting, cells were directly lysed with 2x sodium dodecyl sulfate (SDS) loading buffer (100 mM Tris/HCl, 200 mM dithioerythritol, 4% SDS, 10 mM EDTA, 0.2% bromophenol blue, 20% glycerol). Whole-cell lysates were separated by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes (GE Healthcare). Immunodetection was performed with anti-HA (3F10; Roche), anti-Pes1 (8E9), anti-Bop1 (6H11), anti-WDR12 (1B8), antinucleostemin (7H3), anti-Nog1 (1D8), anti-NPM1 (clone FC82291; Sigma Aldrich), antitubulin (Sigma Aldrich), anti-p53 (PAb240; Dianova), and anti-c-Myc (N-262; Santa Cruz Biotechnology, Inc.). Recombinant mouse Bop1 protein was detected with a 1:10,000 dilution of polyclonal mouse Bop1-specific guinea pig antibodies in methanol-acetone-fixed cells. Immunofluorescence and immunoprecipitation have been described elsewhere (14).
Native gel electrophoresis. Cells (3 x 106) were lysed in 100 µl lysis buffer (50 mM Tris-HCl [pH 8.0], 1% NP-40, 150 mM NaCl, phosphatase inhibitors, protease inhibitors) at 4°C for 20 min. A 7.5-µl volume of 2x sample buffer (125 mM Tris-HCl [pH 6.8], 30% glycerol, 0.02% bromophenol blue) was added to 7.5 µl of total lysate and separated by polyacrylamide gel electrophoresis (6.5%) in the absence of SDS at 4°C. Blotting was performed in the absence of methanol. Immunoblotting was performed as described above.
siRNA transfection.
The day before transfection, 
5 x 104 to 105 cells were seeded in six-well plates. Five microliters of 20 µM control, Pes1-, Bop1-, or WDR12-specific small interfering RNA (siRNA) was diluted in 150 µl of OptiMEM (Invitrogen). One hundred fifty microliters of OptiMEM containing 5 µl of Oligofectamine (Invitrogen) was added, and the mixture was incubated for 15 min. Finally, 600 µl of OptiMEM was added and the mixture was applied to cells after aspiration of the culture medium. Cells were incubated for 5 to 6 h. The following sequences (sense) were used: Pes1 UTR, CCAGAGGACCUAAGUGUGAdTdT; Pes1 ORF, AGGUCUUCCUGUCCAUCAAdTDT; Bop1 UTR, UCGUGCUGAAGUCAACAGAdTdT; Bop1 ORF, AUGGCAUGGUGUACAAUGAdTdT; WDR12 UTR-1, CGUACGUUUCCGUGGGCAAdTdT; WDR12 UTR-2, CGCUUACCUGUGCAGUCUAdTdT; Control (nonspecific siRNA), UUCUCCGAACGUGUCACGUdTdT.
Knockdown-knock-in assay. Exogenous gene expression (Pes1, Bop1, or WDR12) was activated for 3 days by treatment with 1 µg/ml doxycycline and then maintained throughout the subsequent course of two siRNA transfections with siRNAs directed against the 3' untranslated region (UTR) of the Pes1, Bop1, or WDR12 mRNA.
Cell fractionation and sucrose gradients. Cells were harvested by trypsinization and washed three times with cold phosphate-buffered saline. Cells (3 x 106) were lysed in 100 µl lysis buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.5% NP-40, protease inhibitors) at 4°C for 20 min. Cytoplasmic fractions were isolated after centrifugation. The pelleted nuclei were washed three times with cold lysis buffer A and then lysed in 100 µl lysis buffer B (50 mM Tris-HCl [pH 8.0], 1% NP-40, 150 mM NaCl, protease inhibitors) at 4°C for 20 min. Sucrose gradients have been described elsewhere (10).
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
The PeBoW complex is involved in pre-rRNA processing. Therefore, we studied the maturation of rRNA in cells overexpressing individual PeBoW components. A scheme of mammalian RNA-processing pathways is shown in Fig. 1E. Pes1, Bop1, WDR12, and combinations thereof were stably expressed in H1299 and TGR-1 (data not shown) cells for 24 h. Total RNA was isolated and analyzed by Northern blot analysis with probes specific for internal transcribed spacer 1 (ITS-1) and ITS-2 of the ribosomal pre-rRNA (Fig. 1F and G). Bop1-overexpressing cells accumulated the 47/45S and 32S pre-rRNAs by twofold (Fig. 1F and G, lanes 2) whereas overexpression of WDR12 or Pes1 did not interfere with pre-rRNA processing (Fig. 1F and G, lanes 3 and 5). The Bop1-mediated aberrant accumulation of pre-rRNAs could be completely reversed through coexpression of WDR12, but not Pes1 (Fig. 1F and G, lanes 4 and 6). These results are in line with the proliferation experiments showing that coexpression of WDR12 but not Pes1 alleviates the negative effects of Bop1 overexpression.
Overexpression of Bop1 titrates endogenous WDR12 into a Bop1/WDR12 subcomplex. These results prompted us to investigate the impact of Bop1 overexpression on the PeBoW complex in more detail. We examined whether ectopically expressed Pes1, Bop1, or WDR12 affected the abundance of the endogenous PeBoW proteins. Endogenous Pes1 and WDR12 can be discriminated from the human HA-tagged forms by their lower molecular weights. Recombinant rodent HA-Bop1 is not recognized by the human-specific Bop1 MAb. Overexpression of HA-Bop1, but not WDR12-HA, Pes1-HA, or luciferase, strongly reduced the steady-state levels of endogenous Bop1 in H1299 cells after 6 days (Fig. 2A). However, such a decrease in the endogenous Bop1 protein level was not detected in cells overexpressing Bop1 for only 1 day (data not shown). We did not observe a decrease in the protein levels of endogenous proteins WDR12 and Pes1 (Fig. 2A, lanes 2 and 4) after expression of Pes1-HA and WDR12-HA, respectively.
|
To verify that recombinant Bop1 is incorporated into the PeBoW complex, we performed native gel electrophoresis after 6 days of Bop1 overexpression (Fig. 2C). Recombinant Bop1, which is only recognized by the HA-specific but not the human-specific Bop1 MAb, replaced the endogenous Bop1 in PeBoW complex assembly (Fig. 2C, lanes 2 and 4).
As overexpression of Bop1 titrates endogenous WDR12 into a Bop1/WDR12 subcomplex without Pes1, we tested whether additional coexpression of Pes1 restores PeBoW complex formation. U2OS cells were stably transfected with two or three individual pRTS constructs. We performed native gel electrophoresis after 6 days of coexpression. The immunoblot of WDR12 shows that monomeric WDR12 (Fig. 2D, lane 1) builds a subcomplex with coexpressed Bop1 (lane 2) but not with coexpressed Pes1 (lane 3). The recombinant expression of all three PeBoW subunits is sufficient for the establishment of the PeBoW complex (Fig. 2D, lane 4). Equal expression levels were determined by Western blot analysis (Fig. 2D).
Overexpression of Bop1 and Pes1 induces a Bop1/Pes1 subcomplex. Overexpression of Bop1 did not reveal a detectable subcomplex with the endogenous Pes1, possibly because of its low stability. To test whether such a complex can generally be formed, we overexpressed Bop1 and Pes1 together. Cell fractionation experiments revealed a Bop1/Pes1 subcomplex in the nuclear fraction but not in the cytoplasmic fraction (Fig. 2E). In contrast, the Bop1/WDR12 subcomplex appeared only in the cytoplasmic fraction and was absent from the nuclear fraction.
Knockdown of Pes1 induces a Bop1/WDR12 subcomplex. Overexpression of Bop1 resulted in the formation of an incomplete PeBoW complex containing Bop1 and WDR12 but not Pes1. Therefore, we aimed to investigate whether this Bop1/WDR12 subcomplex would also appear in cells depleted of Pes1. We performed endogenous Pes1, Bop1, and WDR12 siRNA knockdown experiments. U2OS cells were transfected two times with siRNAs directed against the UTR or open reading frame of WDR12, Bop1, or Pes1 mRNA. All siRNAs induced a strong reduction of the respective proteins within 2 days after the last transfection (Fig. 3A). We also tested whether knockdown of Pes1, Bop1, or WDR12 interfered with rRNA processing. Metabolic labeling of nascent rRNA revealed that cells depleted of a single PeBoW component failed to produce mature 28S rRNA (Fig. 3B).
|
Bop1 mediates the interaction of Pes1 and WDR12. WDR12 and Bop1, as well as Pes1 and Bop1, can form a subcomplexes, but we failed to detect a Pes1/WDR12 subcomplex. Therefore, we studied the interaction of Pes1, Bop1, and WDR12 in more detail. U2OS cells were transfected two times with either control or Bop1-specific siRNA, and coimmunoprecipitation experiments were performed (Fig. 3D). In control cells, WDR12 and Pes1 were specifically immunoprecipitated and also coimmunoprecipitated (Fig. 3D, lanes 3 to 5). In contrast, WDR12 and Pes1 could not be coimmunoprecipitated in Bop1-depleted cells (Fig. 3D, lanes 8 and 9). Therefore, we suppose that the interaction of Pes1 and WDR12 is indirect and mediated by the core factor Bop1.
Interdependent stability of PeBoW components. Native gel electrophoresis revealed that Pes1-, Bop1-, or WDR12-depleted cells lack intact PeBoW complexes. Therefore, we analyzed whether depletion of single PeBoW components affected the abundance of the other components. H1299 cells were transfected twice with siRNA against the 3' UTR of the Pes1, Bop1, or WDR12 mRNA, and cells were harvested daily. The strongest reduction of the respective proteins was observed 3 days after the last siRNA transfection (Fig. 4A). Knockdown of WDR12 slightly reduced the abundance of Bop1 but not of Pes1. In contrast, depletion of Pes1 resulted in a concomitant loss of Bop1 and WDR12. Furthermore, the knockdown of Bop1 strongly reduced the protein levels of Pes1 and WDR12. The abundances of three other nucleolar proteins, Nog1, nucleostemin, and nucleophosmin (NPM1), were not affected. In addition to their nucleolar localization, Nog1 and NPM1 were found in preribosomal complexes and shown to function in the processing of the 28S rRNA (15, 25, 29). These results indicate that the interdependence of the steady-state levels of individual PeBoW components is specific for and restricted to this subcomplex (Fig. 4B).
|
The endogenous Pes1, Bop1, and WDR12 protein levels were studied in cells depleted of the endogenous protein and additionally expressing either of the HA-tagged PeBoW components (Fig. 5). The endogenous, as well as the exogenous HA-tagged, Pes1 and WDR12 proteins were detected by the MAbs but could be discriminated by their increased molecular weights and slower migration due to the C-terminal HA tag. The MAb specific for human Bop1 did not recognize recombinant mouse Bop1-HA. First of all, overexpression of Bop1-HA down-regulated the endogenous Bop1, as described above (Fig. 5A to D, lanes 3), consistent with our previous results (Fig. 2A). Knockdown of WDR12 slightly reduced endogenous Bop1 protein levels but not endogenous Pes1 protein levels (Fig. 5B). This reduction of Bop1 was rescued upon coexpression of WDR12-HA (Fig. 5B, lane 1), but not Pes1-HA or Bop1-HA (lanes 2 and 3). Depletion of endogenous Pes1 caused a concomitant loss of Bop1 and WDR12 (Fig. 5C) that was alleviated by expression of Pes1-HA (Fig. 5C, lane 2), but not WDR12-HA or Bop1-HA (lanes 1 and 3).
|
Interdependence of PeBoW protein levels is transcription independent. In parallel with the Western blot analysis, mRNA levels of Pes1-, Bop1-, or WDR12-depleted cells were investigated by Northern blot analysis (Fig. 6A and B). The knockdown of Pes1, Bop1, or WDR12 strongly reduced the mRNA levels of the respective genes but did not reduce the mRNA levels of other PeBoW components. We even observed a slight increase in mRNA levels. Therefore, the interdependency of the PeBoW proteins is not caused by regulation of the abundance of their mRNA but suggests a translational or posttranslational mechanism.
|
|
The stability of Bop1-HA was further diminished in Pes1-depleted cells (Fig. 7D, lanes 9 to 12) but not in WDR12-depleted cells (lanes 5 to 8). WDR12-HA protein levels were significantly reduced within 24 h in cells lacking endogenous Bop1 (Fig. 7B, lanes 5 to 8) or Pes1 (lanes 9 to 12), and the stability of Pes1 was dependent on the presence of Bop1 (Fig. 7C, lanes 9 to 12) but not WDR12 (lanes 5 to 8). All of these results are fully in line with the mutual dependency of endogenous PeBoW protein levels observed after siRNA knockdown (Fig. 4). In conclusion, the abundance and stability of Pes1, Bop1, and WDR12 are interdependent.
Nucleolar localization of Bop1 requires Pes1. Bop1 overexpression induces subcomplexes of Bop1/WDR12 and Bop1/Pes1. To study the subcellular distribution of these complexes, we preformed indirect immunofluorescence assays with Bop1-specific antibodies. Recombinantly expressed Pes1 and WDR12 proteins show predominant nucleolar staining (10, 14). In contrast, recombinant Bop1 localized exclusively to the cytoplasm in H1299 and TGR-1 cells (Fig. 8A and B). Only cells expressing minor levels of Bop1 also revealed nucleolar staining (data not shown). The cytoplasmic localization of Bop1 was not affected by coexpression of WDR12. However, coexpression of Pes1 fostered the nucleolar localization of Bop1. These results are in line with the cell fractionation experiments in Fig. 2E and further suggest that the titration of WDR12 into the Bop1/WDR12 subcomplex retains WDR12 in the cytoplasm.
|
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
potently blocks rRNA processing and cell cycle progression. Also, in our experiments, Bop1 elicited a profound cell cycle arrest, in contrast to wild-type Bop1, as revealed by a bromodeoxyuridine-light assay investigating the efficiency of reversible cell cycle arrests (unpublished results). However, the comparison of mock-treated cells with wild-type Bop1- and Bop1-overexpressing cells in long-term proliferation assays revealed an inhibitory effect of Bop1 (1). Similarly, overexpressed Bop1 disturbs rRNA processing, although less severely than Bop1, but significantly compared to that in control cells.
|
In addition, depletion of Pes1 triggered the formation of a Bop1/WDR12 subcomplex. Inadequately high levels of Bop1 likewise sequestered endogenous or recombinant WDR12 into this incomplete PeBoW complex. Apparently, the Bop1/WDR12 subcomplex resulted from a relative deficiency of Pes1, as additionally providing sufficient amounts of Pes1 restored PeBoW formation. Alternatively, overexpression of Bop1 and Pes1 induced the formation of a Bop1/Pes1 subcomplex. Thus, overexpression of Bop1 leads to sequestration of Pes1 and WDR12 in two subcomplexes, each containing Bop1 (Fig. 10). In addition, cell fractionation, indirect immunofluorescence, and sucrose gradient centrifugation experiments demonstrated that the subcomplexes behave differently. While the Bop1/WDR12 subcomplex is retained in the cytoplasm, the Bop1/Pes1 subcomplex is located in the nucleolus (Fig. 10). It is further evident that Bop1 requires the help of Pes1 for translocation into the nucleolus. We could recently show that two domains are essential for the nucleolar transport of Pes1, the Bop1 interaction domain and the BRCT domain (10, 13).
Studies of preribosomal complexes in yeast suggest that the Bop1 and Pes1 homologues Erb1p and Nop7p assemble at the preribosome prior to the WDR12 homologue Ytm1p (2, 22). Overexpressed WDR12 is largely located in the nucleolus (14), suggesting that in mammals nucleolar transport of WDR12 also occurs independently of Pes1 and Bop1 and that the PeBoW complex assembles in the nucleolus. Interdependency of factors has recently also been reported for the activity of the SSU processome in yeast (32).
In the present study, we have identified a crucial role for Bop1 in PeBoW homeostasis. As the gene for Bop1 is frequently amplified in colorectal cancers (18), its deregulation might be involved in carcinogenesis. Overexpression of Bop1, as well as expression of a dominant negative C-terminally truncated Bop protein, affects ribosome biogenesis (28). Whether Bop1 overexpression increases the risk of cancer by impairment of the PeBoW complex or by interaction with other cellular factors like the Cdc14 phosphatase (12), which is important for exit of mitosis, remains unclear and deserves further investigation. We also cannot rule out the possibility that overexpression of Bop1 induces p53 and thereby selects for mutants inactivating p53.
We have conclusively shown that the PeBoW complex is essential for ribosome biogenesis and that its integrity is controlled by its interdependent subunits Pes1, Bop1, and WDR12. The PeBoW complex demonstrates that the stability of free and complex-associated proteins can differ substantially. This complex-specific mutual dependency of protein stability might serve as a control mechanism to accurately adjust expression levels and to ensure correct subcomplex assembly, subsequently required for the proper maturation of a large macromolecule such as the ribosome.
|
ACKNOWLEDGMENTS |
|---|
This work was supported by the Deutsche Forschungsgemeinschaft (EI 216/8-1, SFB684, SFB-Transregio5).
We have no conflict of interest.
|
FOOTNOTES |
|---|
Published ahead of print on 12 March 2007.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2. Dez, C., and D. Tollervey. 2004. Ribosome synthesis meets the cell cycle. Curr. Opin. Microbiol. 7:631-637.[CrossRef][Medline]
3. Dosil, M., and X. R. Bustelo. 2004. Functional characterization of Pwp2, a WD family protein essential for the assembly of the 90S pre-ribosomal particle. J. Biol. Chem. 279:37385-37397.
4. Dragon, F., J. E. Gallagher, P. A. Compagnone-Post, B. M. Mitchell, K. A. Porwancher, K. A. Wehner, S. Wormsley, R. E. Settlage, J. Shabanowitz, Y. Osheim, A. L. Beyer, D. F. Hunt, and S. J. Baserga. 2002. A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. Nature 417:967-970.[CrossRef][Medline]
5. Eichler, D. C., and N. Craig. 1994. Processing of eukaryotic ribosomal RNA. Prog. Nucleic Acid Res. Mol. Biol. 49:197-239.[Medline]
6. Fatica, A., and D. Tollervey. 2002. Making ribosomes. Curr. Opin. Cell Biol. 14:313-318.[CrossRef][Medline]
7. Fontoura, B. M., C. A. Atienza, E. A. Sorokina, T. Morimoto, and R. B. Carroll. 1997. Cytoplasmic p53 polypeptide is associated with ribosomes. Mol. Cell. Biol. 17:3146-3154.[Abstract]
8. Fromont-Racine, M., B. Senger, C. Saveanu, and F. Fasiolo. 2003. Ribosome assembly in eukaryotes. Gene 313:17-42.[CrossRef][Medline]
9. Gallagher, J. E., D. A. Dunbar, S. Granneman, B. M. Mitchell, Y. Osheim, A. L. Beyer, and S. J. Baserga. 2004. RNA polymerase I transcription and pre-rRNA processing are linked by specific SSU processome components. Genes Dev. 18:2506-2517.
10. Grimm, T., M. Hölzel, M. Rohrmoser, T. Harasim, A. Malamoussi, A. Gruber-Eber, E. Kremmer, and D. Eick. 2006. Dominant-negative Pes1 mutants inhibit ribosomal RNA processing and cell proliferation via incorporation into the PeBoW-complex. Nucleic Acids Res. 34:3030-3043.
11. Harnpicharnchai, P., J. Jakovljevic, E. Horsey, T. Miles, J. Roman, M. Rout, D. Meagher, B. Imai, Y. Guo, C. J. Brame, J. Shabanowitz, D. F. Hunt, and J. L. Woolford, Jr. 2001. Composition and functional characterization of yeast 66S ribosome assembly intermediates. Mol. Cell 8:505-515.[CrossRef][Medline]
12. Ho, Y., A. Gruhler, A. Heilbut, G. D. Bader, L. Moore, S. L. Adams, A. Millar, P. Taylor, K. Bennett, K. Boutilier, L. Yang, C. Wolting, I. Donaldson, S. Schandorff, J. Shewnarane, M. Vo, J. Taggart, M. Goudreault, B. Muskat, C. Alfarano, D. Dewar, Z. Lin, K. Michalickova, A. R. Willems, H. Sassi, P. A. Nielsen, K. J. Rasmussen, J. R. Andersen, L. E. Johansen, L. H. Hansen, H. Jespersen, A. Podtelejnikov, E. Nielsen, J. Crawford, V. Poulsen, B. D. Sorensen, J. Matthiesen, R. C. Hendrickson, F. Gleeson, T. Pawson, M. F. Moran, D. Durocher, M. Mann, C. W. Hogue, D. Figeys, and M. Tyers. 2002. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415:180-183.[CrossRef][Medline]
13. Hölzel, M., T. Grimm, M. Rohrmoser, A. Malamoussi, T. Harasim, A. Gruber-Eber, E. Kremmer, and D. Eick. 2007. The BRCT domain of mammalian Pes1 is crucial for nucleolar localization and rRNA processing. Nucleic Acids Res. 35:789-800.
14. Hölzel, M., M. Rohrmoser, M. Schlee, T. Grimm, T. Harasim, A. Malamoussi, A. Gruber-Eber, E. Kremmer, W. Hiddemann, G. W. Bornkamm, and D. Eick. 2005. Mammalian WDR12 is a novel member of the Pes1-Bop1 complex and is required for ribosome biogenesis and cell proliferation. J. Cell Biol. 170:367-378.
15. Jensen, B. C., Q. Wang, C. T. Kifer, and M. Parsons. 2003. The NOG1 GTP-binding protein is required for biogenesis of the 60S ribosomal subunit. J. Biol. Chem. 278:32204-32211.
16. Jorgensen, P., and M. Tyers. 2004. How cells coordinate growth and division. Curr. Biol. 14:R1014-R1027.[CrossRef][Medline]
17. Killian, A., N. Le Meur, R. Sesboue, J. Bourguignon, G. Bougeard, J. Gautherot, C. Bastard, T. Frebourg, and J. M. Flaman. 2004. Inactivation of the RRB1-Pescadillo pathway involved in ribosome biogenesis induces chromosomal instability. Oncogene 23:8597-8602.[CrossRef][Medline]
18. Killian, A., N. Sarafan-Vasseur, R. Sesboue, F. Le Pessot, F. Blanchard, A. Lamy, M. Laurent, J. M. Flaman, and T. Frebourg. 2006. Contribution of the BOP1 gene, located on 8q24, to colorectal tumorigenesis. Genes Chromosomes Cancer 45:874-881.[CrossRef][Medline]
19. Krogan, N. J., W. T. Peng, G. Cagney, M. D. Robinson, R. Haw, G. Zhong, X. Guo, X. Zhang, V. Canadien, D. P. Richards, B. K. Beattie, A. Lalev, W. Zhang, A. P. Davierwala, S. Mnaimneh, A. Starostine, A. P. Tikuisis, J. Grigull, N. Datta, J. E. Bray, T. R. Hughes, A. Emili, and J. F. Greenblatt. 2004. High-definition macromolecular composition of yeast RNA-processing complexes. Mol. Cell 13:225-239.[CrossRef][Medline]
20. Lapik, Y. R., C. J. Fernandes, L. F. Lau, and D. G. Pestov. 2004. Physical and functional interaction between Pes1 and Bop1 in mammalian ribosome biogenesis. Mol. Cell 15:17-29.[CrossRef][Medline]
21. Lohrum, M. A., R. L. Ludwig, M. H. Kubbutat, M. Hanlon, and K. H. Vousden. 2003. Regulation of HDM2 activity by the ribosomal protein L11. Cancer Cell 3:577-587.[CrossRef][Medline]
22. Miles, T. D., J. Jakovljevic, E. W. Horsey, P. Harnpicharnchai, L. Tang, and J. L. Woolford, Jr. 2005. Ytm1, Nop7, and Erb1 form a complex necessary for maturation of yeast 66S preribosomes. Mol. Cell. Biol. 25:10419-10432.
23. Pestov, D. G., Z. Strezoska, and L. F. Lau. 2001. Evidence of p53-dependent cross-talk between ribosome biogenesis and the cell cycle: effects of nucleolar protein Bop1 on G1/S transition. Mol. Cell. Biol. 21:4246-4255.
24. Rudra, D., and J. R. Warner. 2004. What better measure than ribosome synthesis? Genes Dev. 18:2431-2436.
25. Savkur, R. S., and M. O. Olson. 1998. Preferential cleavage in pre-ribosomal RNA byprotein B23 endoribonuclease. Nucleic Acids Res. 26:4508-4515.
26. Schlosser, I., M. Hölzel, M. Murnseer, H. Burtscher, U. H. Weidle, and D. Eick. 2003. A role for c-Myc in the regulation of ribosomal RNA processing. Nucleic Acids Res. 31:6148-6156.
27. Sherr, C. J., and J. D. Weber. 2000. The ARF/p53 pathway. Curr. Opin. Genet. Dev. 10:94-99.[CrossRef][Medline]
28. Strezoska, Z., D. G. Pestov, and L. F. Lau. 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.
29. Takahashi, N., M. Yanagida, S. Fujiyama, T. Hayano, and T. Isobe. 2003. Proteomic snapshot analyses of preribosomal ribonucleoprotein complexes formed at various stages of ribosome biogenesis in yeast and mammalian cells. Mass Spectrom. Rev. 22:287-317.[CrossRef][Medline]
30. Thiry, M., and D. L. Lafontaine. 2005. Birth of a nucleolus: the evolution of nucleolar compartments. Trends Cell Biol. 15:194-199.[CrossRef][Medline]
31. Warner, J. R. 1999. The economics of ribosome biosynthesis in yeast. Trends Biochem. Sci. 24:437-440.[CrossRef][Medline]
32. Wehner, K. A., J. E. Gallagher, and S. J. Baserga. 2002. Components of an interdependent unit within the SSU processome regulate and mediate its activity. Mol. Cell. Biol. 22:7258-7267.
33. Zhang, Y., G. W. Wolf, K. Bhat, A. Jin, T. Allio, W. A. Burkhart, and Y. Xiong. 2003. Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway. Mol. Cell. Biol. 23:8902-8912.
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|
-tubulin. (C) The proliferation of stable polyclonal H1299 cell lines expressing the indicated proteins was analyzed as described for panel A. (D) Expression levels of HA-tagged proteins were determined as described for panel B. (E) Diagram of the primary 47S rRNA transcript and the major rRNA intermediates. Positions of the hybridization probes are depicted. ETS, external transcribed spacer. (F) Northern blot analysis of rRNA precursors. Total RNA was extracted from subconfluent H1299 cells expressing the indicated HA-tagged proteins for 24 h. Equal amounts of total RNA were separated by agarose-formaldehyde gel electrophoresis and hybridized with probes specific for ITS-1 and ITS-2 of the rRNA intermediates. As a loading control, blots were incubated with a probe specific for 18S rRNA. (G) Ratios of 47S-45S/18S and 32S/18S rRNAs.
