The Nucleolar Protein Myb-binding Protein 1A (MYBBP1A) Enhances p53 Tetramerization and Acetylation in Response to Nucleolar Disruption

Tetramerization of p53 is crucial to exert its biological activity, and nucleolar disruption is sufficient to activate p53. We previously demonstrated that nucleolar stress induces translocation of the nucleolar protein MYBBP1A from the nucleolus to the nucleoplasm and enhances p53 activity. However, whether and how MYBBP1A regulates p53 tetramerization in response to nucleolar stress remain unclear. In this study, we demonstrated that MYBBP1A enhances p53 tetramerization, followed by acetylation under nucleolar stress. We found that MYBBP1A has two regions that directly bind to lysine residues of the p53 C-terminal regulatory domain. MYBBP1A formed a self-assembled complex that provided a molecular platform for p53 tetramerization and enhanced p300-mediated acetylation of the p53 tetramer. Moreover, our results show that MYBBP1A functions to enhance p53 tetramerization that is necessary for p53 activation, followed by cell death with actinomycin D treatment. Thus, we suggest that MYBBP1A plays a pivotal role in the cellular stress response.     

Critical role of the nucleolus in activation of the p53-dependent postmitotic checkpoint

Cells eventually exit from mitosis during sustained arrest at the spindle checkpoint, without sister chromatid separation and cytokinesis. The resulting tetraploid cells are arrested in the subsequent G1 phase in a p53-dependent manner by the regulatory function of the postmitotic G1 checkpoint. Here we report how the nucleolus plays a critical role in activation of the postmitotic G1 checkpoint. During mitosis, the nucleolus is disrupted and many nucleolar proteins are translocated from the nucleolus into the cytoplasm. Among the nucleolar factors, Myb-binding protein 1a (MYBBP1A) induces the acetylation and accumulation of p53 by enhancing the interaction between p300 and p53 during prolonged mitosis. MYBBP1A-dependent p53 activation is essential for the postmitotic G1 checkpoint. Thus, our results demonstrate a novel nucleolar function that monitors the prolongation of mitosis and converts its signal into activation of the checkpoint machinery.     

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.     

Mutations in nucleolar proteins lead to nucleolar accumulation of polyA+ RNA in Saccharomyces cerevisiae.

Synthesis of mRNA and rRNA occur in the chromatin-rich nucleoplasm and the nucleolus, respectively. Nevertheless, we here report that a Saccharomyces cerevisiae gene, MTR3, previously implicated in mRNA transport, codes for a novel essential 28-kDa nucleolar protein. Moreover, in mtr3-1 the accumulated polyA+ RNA actually colocalizes with nucleolar antigens, the nucleolus becomes somewhat disorganized, and rRNA synthesis and processing are inhibited. A strain with a ts conditional mutation in RNA polymerase I also shows nucleolar accumulation of polyA+ RNA, whereas strains with mutations in the nucleolar protein Nop1p do not. Thus, in several mutant backgrounds, when mRNA cannot be exported i concentrates in the nucleolus. mRNA may normally encounter nucleolar components before export and proteins such as Mtr3p may be critical for export of both mRNA and ribosomal subunits.     

GRWD1 negatively regulates p53 via the RPL11–MDM2 pathway and promotes tumorigenesis

The ribosomal protein L11 (RPL11) binds and inhibits the MDM2 ubiquitin ligase, thereby promoting p53 stability. Thus, RPL11 acts as a tumor suppressor. Here, we show that GRWD1 (glutamate‐rich WD40 repeat containing 1) physically and functionally interacts with RPL11. GRWD1 is localized to nucleoli and is released into the nucleoplasm upon nucleolar stress. Silencing of GRWD1 increases p53 induction by nucleolar stress, whereas overexpression of GRWD1 reduces p53 induction. Furthermore, GRWD1 overexpression competitively inhibits the RPL11–MDM2 interaction and alleviates RPL11‐mediated suppression of MDM2 ubiquitin ligase activity toward p53. These effects are mediated by the N‐terminal region of GRWD1, including the acidic domain. Finally, we show that GRWD1 overexpression in combination with HPV16 E7 and activated KRAS confers anchorage‐independent growth and tumorigenic capacity on normal human fibroblasts. Consistent with this, GRWD1 overexpression is associated with poor prognosis in cancer patients. Taken together, our results suggest that GRWD1 is a novel negative regulator of p53 and a potential oncogene.     

Silencing of Ribosomal Protein S9 Elicits a Multitude of Cellular Responses Inhibiting the Growth of Cancer Cells Subsequent to p53 Activation

Background

Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9).

Methodology/Principal Findings

Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis.

Conclusions

p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.     

GRWD1, a new player among oncogenesis-related ribosomal/nucleolar proteins

Increasing attention has been paid to certain ribosomal or ribosome biosynthesis-related proteins involved in oncogenesis. Members of one group are classified as “tumor suppressive factors” represented by RPL5 and RPL11; loss of their functions leads to cancer predisposition. RPL5 and RPL11 prevent tumorigenesis by binding to and inhibiting the MDM2 ubiquitin ligase and thereby up-regulating p53. Many other candidate tumor suppressive ribosomal/nucleolar proteins have been suggested. However, it remains to be experimentally clarified whether many of these factors can actually prevent tumorigenesis and if so, how they do so. Conversely, some ribosomal/nucleolar proteins promote tumorigenesis. For example, PICT1 binds to and anchors RPL11 in nucleoli, down-regulating p53 and promoting tumorigenesis. GRWD1 was recently identified as another such factor. When overexpressed, GRWD1 suppresses p53 and transforms normal human cells, probably by binding to RPL11 and sequestrating it from MDM2. However, other pathways may also be involved.     

Interactions of nucleolin and ribosomal protein L26 (RPL26) in translational control of human p53 mRNA

Ribosomal protein RPL26 enhances p53 translation after DNA damage, and this regulation depends upon interactions between the 5'- and 3'-UTRs of human p53 mRNA (Takagi, M., Absalon, M. J., McLure, K. G., and Kastan, M. B. (2005) Cell 123, 49-63; Chen, J., and Kastan, M. B. (2010) Genes Dev. 24, 2146-2156). In contrast, nucleolin (NCL) suppresses the translation of p53 mRNA and its induction after DNA damage. We confirmed reports that RPL26 and NCL interact with each other and then explored the potential role of this interaction in the translational control of p53 after stress. NCL repression of p53 translation utilizes both the 5'- and 3'-UTRs of p53 mRNA, and NCL binds to the same 5'-3'-UTR interaction region that is critical for the recruitment of RPL26 to p53 mRNA after DNA damage. We also found that NCL is able to oligomerize, consistent with a model in which NCL stabilizes this double-stranded RNA structure. We found that the RNA-binding domain of NCL participates in binding to p53 mRNA, is required for both NCL dimerization and NCL-mediated translational repression, and is the domain of NCL that interacts with RPL26. Excessive RPL26 disrupts NCL dimerization, and point mutations in the NCL-interacting region of RPL26 reduce NCL-RPL26 interactions and attenuate both RPL26 binding to human p53 mRNA and p53 induction by RPL26. These observations suggest a model in which the base pairings in the p53 UTR interaction regions are critical for both translational repression and stress induction of p53 by NCL and RPL26, respectively, and that disruption of a NCL-NCL homodimer by RPL26 may be the switch between translational repression and activation after stress.