Ribosomal protein S9 is a novel B23/NPM-binding protein required for normal cell proliferation

B23 (NPM/nucleophosmin) is a multifunctional nucleolar protein and a member of the nucleoplasmin superfamily of acidic histone chaperones. B23 is essential for normal embryonic development and plays an important role in genomic stability, ribosome biogenesis, and anti-apoptotic signaling. Altered protein expression or genomic mutation of B23 is encountered in many different forms of cancer. Although described as multifunctional, a genuine molecular function of B23 is not fully understood. Here we show that B23 is associated with a protein complex consisting of ribosomal proteins and ribosome-associated RNA helicases. A novel, RNA-independent interaction between ribosomal protein S9 (RPS9) and B23 was further investigated. We found that S9 binding requires an intact B23 oligomerization domain. Depletion of S9 by small interfering RNA resulted in decreased protein synthesis and G(1) cell cycle arrest, in association with induction of p53 target genes. We determined that S9 is a short-lived protein in the absence of ribosome biogenesis, and proteasomal inhibition significantly increased S9 protein level. Overexpression of B23 facilitated nucleolar storage of S9, whereas knockdown of B23 led to diminished levels of nucleolar S9. Our results suggest that B23 selectively stores, and protects ribosomal protein S9 in nucleoli and therefore could facilitate ribosome biogenesis.     

Perturbation of RNA Polymerase I transcription machinery by ablation of HEATR1 triggers the RPL5/RPL11-MDM2-p53 ribosome biogenesis stress checkpoint pathway in human cells

Ribosome biogenesis is an energy consuming process which takes place mainly in the nucleolus. By producing ribosomes to fuel protein synthesis, it is tightly connected with cell growth and cell cycle control. Perturbation of ribosome biogenesis leads to the activation of p53 tumor suppressor protein promoting processes like cell cycle arrest, apoptosis or senescence. This ribosome biogenesis stress pathway activates p53 through sequestration of MDM2 by a subset of ribosomal proteins (RPs), thereby stabilizing p53. Here, we identify human HEATR1, as a nucleolar protein which positively regulates ribosomal RNA (rRNA) synthesis. Downregulation of HEATR1 resulted in cell cycle arrest in a manner dependent on p53. Moreover, depletion of HEATR1 also caused disruption of nucleolar structure and activated the ribosomal biogenesis stress pathway – RPL5 / RPL11 dependent stabilization and activation of p53. These findings reveal an important role for HEATR1 in ribosome biogenesis and further support the concept that perturbation of ribosome biosynthesis results in p53-dependent cell cycle checkpoint activation, with implications for human pathologies including cancer.     

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

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.     

DOMINO1, a member of a small plant-specific gene family, encodes a protein essential for nuclear and nucleolar functions

Arabidopsis embryos carrying the domino1 mutation grow slowly in comparison with wild type embryos and as a consequence reach only the globular stage at desiccation. The primary defect of the mutation at the cellular level is the large size of the nucleolus that can be observed soon after fertilization in the nuclei of both the embryo and the endosperm. The ultrastructure of mutant nucleoli is drastically different from wild type and points to a fault in ribosome biogenesis. DOMINO1 encodes a protein, which belongs to a plant-specific gene family sharing a common motif of unknown function, present in the tomato DEFECTIVE CHLOROPLASTS AND LEAVES (LeDCL) protein. Using a GFP protein fusion, we show that DOMINO1 is targeted to the nucleus. We propose that inactivation of DOMINO1 has a negative effect on ribosome biogenesis and on the rate of cell division.     

Tumor suppressor ARF degrades B23, a nucleolar protein involved in ribosome biogenesis and cell proliferation

The tumor suppressor ARF induces a p53-dependent and -independent cell cycle arrest. Unlike the nucleoplasmic MDM2 and p53, ARF localizes in the nucleolus. The role of ARF in the nucleolus, the molecular target, and the mechanism of its p53-independent function remains unclear. Here we show that ARF interacts with B23, a multifunctional nucleolar protein involved in ribosome biogenesis, and promotes its polyubiquitination and degradation. Overexpression of B23 induces a cell cycle arrest in normal fibroblasts, whereas in cells lacking p53 it promotes S phase entry. Conversely, knocking down B23 inhibits the processing of preribosomal RNA and induces cell death. Further, oncogenic Ras induces B23 only in ARF null cells, but not in cells that retain wild-type ARF. Together, our results reveal a molecular mechanism of ARF in regulating ribosome biogenesis and cell proliferation via inhibiting B23, and suggest a nucleolar role of ARF in surveillance of oncogenic insults.     

NO66, a highly conserved dual location protein in the nucleolus and in a special type of synchronously replicating chromatin

It has recently become clear that the nucleolus, the most prominent nuclear subcompartment, harbors diverse functions beyond its classic role in ribosome biogenesis. To gain insight into nucleolar functions, we have purified amplified nucleoli from Xenopus laevis oocytes using a novel approach involving fluorescence-activated cell sorting techniques. The resulting protein fraction was analyzed by mass spectrometry and used for the generation of monoclonal antibodies directed against nucleolar components. Here, we report the identification and molecular characterization of a novel, ubiquitous protein, which in most cell types appears to be a constitutive nucleolar component. Immunolocalization studies have revealed that this protein, termed NO66, is highly conserved during evolution and shows in most cells analyzed a dual localization pattern, i.e., a strong enrichment in the granular part of nucleoli and in distinct nucleoplasmic entities. Colocalizations with proteins Ki-67, HP1alpha, and PCNA, respectively, have further shown that the staining pattern of NO66 overlaps with certain clusters of late replicating chromatin. Biochemical experiments have revealed that protein NO66 cofractionates with large preribosomal particles but is absent from cytoplasmic ribosomes. We propose that in addition to its role in ribosome biogenesis protein NO66 has functions in the replication or remodeling of certain heterochromatic regions.     

Physical and functional interaction between Pes1 and Bop1 in mammalian ribosome biogenesis

Molecular mechanisms of mammalian ribosome biogenesis remain largely unexplored. Here we develop a series of transposon-derived dominant mutants of Pes1, the mouse homolog of the zebrafish Pescadillo and yeast Nop7p implicated in ribosome biogenesis and cell proliferation control. Six Pes1 mutants selected by their ability to reversibly arrest the cell cycle also impair maturation of the 28S and 5.8S rRNAs in mouse cells. We show that Pes1 physically interacts with the nucleolar protein Bop1, and both proteins direct common pre-rRNA processing steps. Interaction with Bop1 is essential for the efficient incorporation of Pes1 into nucleolar preribosomal complexes. Pes1 mutants defective for the interaction with Bop1 lose the ability to affect rRNA maturation and the cell cycle. These data show that coordinated action of Pes1 and Bop1 is necessary for the biogenesis of 60S ribosomal subunits.     

Evidence of p53-dependent cross-talk between ribosome biogenesis and the cell cycle: effects of nucleolar protein Bop1 on G(1)/S transition

Bop1 is a novel nucleolar protein involved in rRNA processing and ribosome assembly. We have previously shown that expression of Bop1Delta, an amino-terminally truncated Bop1 that acts as a dominant negative mutant in mouse cells, results in inhibition of 28S and 5.8S rRNA formation and deficiency of newly synthesized 60S ribosomal subunits (Z. Strezoska, D. G. Pestov, and L. F. Lau, Mol. Cell. Biol. 20:5516-5528, 2000). Perturbation of Bop1 activities by Bop1Delta also induces a powerful yet reversible cell cycle arrest in 3T3 fibroblasts. In the present study, we show that asynchronously growing cells are arrested by Bop1Delta in a highly concerted fashion in the G(1) phase. Kinase activities of the G(1)-specific Cdk2 and Cdk4 complexes were downregulated in cells expressing Bop1Delta, whereas levels of the Cdk inhibitors p21 and p27 were concomitantly increased. The cells also displayed lack of hyperphosphorylation of retinoblastoma protein (pRb) and decreased expression of cyclin A, indicating their inability to progress through the restriction point. Inactivation of functional p53 abrogated this Bop1Delta-induced cell cycle arrest but did not restore normal rRNA processing. These findings show that deficiencies in ribosome synthesis can be uncoupled from cell cycle arrest and reveal a new role for the p53 pathway as a mediator of the signaling link between ribosome biogenesis and the cell cycle. We propose that aberrant rRNA processing and/or ribosome biogenesis may cause "nucleolar stress," leading to cell cycle arrest in a p53-dependent manner.     

A small ribosomal subunit (SSU) processome component, the human U3 protein 14A (hUTP14A) binds p53 and promotes p53 degradation

Ribosome biogenesis is required for normal cell function, and aberrant ribosome biogenesis can lead to p53 activation. However, how p53 is activated by defects of ribosome biogenesis remains to be determined. Here, we identified human UTP14a as an SSU processome component by showing that hUTP14a is nucleolar, associated with U3 snoRNA and involved in 18 S rRNA processing. Interestingly, ectopic expression of hUTP14a resulted in a decrease and knockdown of hUTP14a led to an increase of p53 protein levels. We showed that hUTP14a physically interacts with p53 and functionally promotes p53 turn-over, and that hUTP14a promotion of p53 destabilization is sensitive to a proteasome inhibitor but independent of ubiquitination. Significantly, knockdown of hUTP14a led to cell cycle arrest and apoptosis. Our data identified a novel pathway for p53 activation through a defect in rRNA processing and suggest that a ribosome biogenesis factor itself could act as a sensor for nucleolar stress to regulate p53.     

The yeast kinase Swe1 is required for proper entry into cell cycle after arrest due to ribosome biogenesis and protein synthesis defects

Sda1 is an essential protein required for cell cycle progression in Saccharomyces cerevisiae. Here, we show that the sda1-1 mutation causes a defect in the formation and nuclear export of 60S ribosomal subunits. Moreover, the sda1-1, but also other mutants defective in ribosome biogenesis (e.g., rix1-1 and tif6Delta), exhibit a G1 arrest, which could be the consequence of impaired ribosome biogenesis. Interestingly, additional deletion of the non-essential Swe1 kinase, the homolog of S. pombe Wee1, causes a pronounced delay in entering a new cell cycle in sda1-1, rix1-1 and tif6Delta cells, when shifted back from restrictive to permissive conditions. However, such a prolonged delay is independent of the Tyr19 phosphorylation in Cdc28. Moreover, the lack of Swe1 causes delay in budding and DNA replication in cells released from the G1 arrest due to the block of protein synthesis. Our data suggest that Swe1 is required for timely entry into cell cycle after a G1 arrest caused by impairment in pre-60S biogenesis and in protein synthesis. Therefore we propose that Swe1, which is required for coordination of cell growth and cell division in G2/M, also has a role in the beginning of the cell cycle.     

The Yeast Kinase Swe1 is Required for Proper Entry into Cell Cycle After Arrest Due to Ribosome Biogenesis and Protein Synthesis Defects

Sda1 is an essential protein required for cell cycle progression in Saccharomyces cerevisiae. Here, we show that the sda1-1 mutation causes a defect in the formation and nuclear export of 60S ribosomal subunits. Moreover, the sda1-1, but also other mutants defective in ribosome biogenesis (e.g., rix1-1 and tif6D), exhibit a G1 arrest, which could be the consequence of impaired ribosome biogenesis. Interestingly, additional deletion of the non-essential Swe1 kinase, the homolog of S. pombe Wee1, causes a pronounced delay in entering a new cell cycle in sda1-1, rix1-1 and tif6D cells, when shifted back from restrictive to permissive conditions. However, such a prolonged delay is independent of the Tyr19 phosphorylation in Cdc28. Moreover, the lack of Swe1 causes delay in budding and DNA replication in cells released from the G1 arrest due to the block of protein synthesis. Our data suggest that Swe1 is required for timely entry into cell cycle after a G1 arrest caused by impairment in pre-60S biogenesis and in protein synthesis. Therefore we propose that Swe1, which is required for coordination of cell growth and cell division in G2/M, also has a role in the beginning of the cell cycle.     

Pescadillo, a novel cell cycle regulatory protein abnormally expressed in malignant cells

Using a culture model of glial tumorigenesis, we identified a novel gene that was up-regulated in malignant mouse astrocytes following the loss of p53. The gene represents the murine homologue of pescadillo, an uncharacterized gene that is essential for embryonic development in zebrafish. Pescadillo is a strongly conserved gene containing unique structural motifs such as a BRCA1 C-terminal domain, clusters of acidic amino acids and consensus motifs for post-translational modification by SUMO-1. Pescadillo displayed a distinct spatial and temporal pattern of gene expression during brain development, being detected in neural progenitor cells and postmitotic neurons. Although it is not expressed in differentiated astrocytes in vivo, the pescadillo protein is dramatically elevated in malignant human astrocytomas. Yeast strains harboring temperature-sensitive mutations in the pescadillo gene were arrested in either G(1) or G(2) when grown in nonpermissive conditions, demonstrating that pescadillo is an essential gene in yeast and is required for cell cycle progression. Consistent with the latter finding, DNA synthesis was only observed in mammalian cells expressing the pescadillo protein. These results suggest that pescadillo plays a crucial role in cell proliferation and may be necessary for oncogenic transformation and tumor progression.     

Loss of Tumor Suppressor RPL5/RPL11 Does Not Induce Cell Cycle Arrest but Impedes Proliferation Due to Reduced Ribosome Content and Translation Capacity

Humans have evolved elaborate mechanisms to activate p53 in response to insults that lead to cancer, including the binding and inhibition of Hdm2 by the 60S ribosomal proteins (RPs) RPL5 and RPL11. This same mechanism appears to be activated upon impaired ribosome biogenesis, a risk factor for cancer initiation. As loss of RPL5/RPL11 abrogates ribosome biogenesis and protein synthesis to the same extent as loss of other essential 60S RPs, we reasoned the loss of RPL5 and RPL11 would induce a p53-independent cell cycle checkpoint. Unexpectedly, we found that their depletion in primary human lung fibroblasts failed to induce cell cycle arrest but strongly suppressed cell cycle progression. We show that the effects on cell cycle progression stemmed from reduced ribosome content and translational capacity, which suppressed the accumulation of cyclins at the translational level. Thus, unlike other tumor suppressors, RPL5/RPL11 play an essential role in normal cell proliferation, a function cells have evolved to rely on in lieu of a cell cycle checkpoint.     

Functions and substrates of NEDDylation during cell cycle in the silkworm,Bombyx mori

NEDDylation, a post-translational modification mediated by the conjugation of the ubiquitin-like protein Nedd8 to specific substrates, is an essential biological process that regulates cell cycle progression in eukaryotes. Here, we report the conservation of NEDDylation machinery and NEDDylated proteins in the silkworm, Bombyx mori. We have identified all the components necessary for reversible NEDDylation in the silkworm including Nedd8, E1, E2, E3, and deNEDDylation enzymes. By the approach of RNAi-mediated gene silencing, it was shown that knockdown of BmNedd8 and the conjugating enzymes decreased the global level of NEDDylation, while knockdown of deNEDDylation enzymes increased the prevalence of this modification in cultured silkworm cells. Moreover, the lack of the NEDDylation system caused cell cycle arrest at the G2/M phase and resulted in defects in chromosome congression and segregation. Using the wild-type and mutants of BmNedd8, we identified the specific substrates of BmNedd8, which are involved in the regulation for many cellular processes, including ribosome biogenesis, spliceosome structure, spindle formation, metabolism, and RNA biogenesis. This clearly demonstrates that the NEDDylation system is able to control multiple pathways in the silkworm. Altogether, the information on the functions and substrates of the NEDDylation system presented here could provide a basis for future investigations of protein NEDDylation and its regulatory mechanism on cell cycle progression in the silkworm.