Regulation of nucleolar signalling to p53 through NEDDylation of L11

Several studies have shown that ribosomal proteins (RPs) are important mediators of p53 activation in response to nucleolar disruption; however, the pathways that control this signalling function of RPs are currently unknown. We have recently shown that RPs are targets for the ubiquitin‐like molecule NEDD8, and that NEDDylation protects RPs from destabilization. Here, we identify NEDD8 as a crucial regulator of L11 RP signalling to p53. A decrease in L11 NEDDylation during nucleolar stress causes relocalization of L11 from the nucleolus to the nucleoplasm. This not only provides the signal for p53 activation, but also makes L11 susceptible to degradation. Mouse double minute 2 (MDM2) ‐mediated NEDDylation protects L11 from degradation and this is required for p53 stabilization during nucleolar stress. By controlling the correct localization and stability of L11, NEDD8 acts as a crucial, new regulator of nucleolar signalling to p53.     

5-fluorouracil activation of p53 involves an MDM2-ribosomal protein interaction

5-fluorouracil (5-FU) is a widely used chemotherapeutic drug for the treatment of a variety of solid tumors. The anti-tumor activity of 5-FU has been attributed in part to its ability to induce p53-dependent cell growth arrest and apoptosis. However, the molecular mechanisms underlying p53 activation by 5-FU remain largely obscure. Here we report that 5-FU treatment leads to p53 stabilization and activation by blocking MDM2 feedback inhibition through ribosomal proteins. 5-FU treatment increased the fraction of ribosome-free L5, L11, and L23 ribosomal proteins and their interaction with MDM2, leading to p53 activation and G1/S arrest. Conversely, individual knockdown of these ribosomal proteins by small interfering RNA prevented the 5-FU-induced p53 activation and reversed the 5-FU-induced G1/S arrest. These results demonstrate that 5-FU treatment triggers a ribosomal stress response so that ribosomal proteins L5, L11, and L23 are released from ribosome to activate p53 by ablating the MDM2-p53 feedback circuit.     

Regulation of the MDM2-p53 pathway by ribosomal protein L11 involves a post-ubiquitination mechanism

Inhibition of the MDM2-p53 feedback loop is critical for p53 activation in response to cellular stresses. The ribosomal proteins L5, L11, and L23 can block this loop by inhibiting MDM2-mediated p53 ubiquitination and degradation in response to ribosomal stress. Here, we show that L11, but not L5 and L23, leads to a drastic accumulation of ubiquitinated and native MDM2. This effect is dependent on the ubiquitin ligase activity of MDM2, but not p53, and requires the central MDM2 binding domain (residues 51-108) of L11. We further show that L11 inhibited 26 S proteasome-mediated degradation of ubiquitinated MDM2 in vitro and consistently prolonged the half-life of MDM2 in cells. These results suggest that L11, unlike L5 and L23, differentially regulates the levels of ubiquitinated p53 and MDM2 and inhibits the turnover and activity of MDM2 through a post-ubiquitination mechanism.     

Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway

The gene encoding p53 mediates a major tumor suppression pathway that is frequently altered in human cancers. p53 function is kept at a low level during normal cell growth and is activated in response to various cellular stresses. The MDM2 oncoprotein plays a key role in negatively regulating p53 activity by either direct repression of p53 transactivation activity in the nucleus or promotion of p53 degradation in the cytoplasm. DNA damage and oncogenic insults, the two best-characterized p53-dependent checkpoint pathways, both activate p53 through inhibition of MDM2. Here we report that the human homologue of MDM2, HDM2, binds to ribosomal protein L11. L11 binds a central region in HDM2 that is distinct from the ARF binding site. We show that the functional consequence of L11-HDM2 association, like that with ARF, results in the prevention of HDM2-mediated p53 ubiquitination and degradation, subsequently restoring p53-mediated transactivation, accumulating p21 protein levels, and inducing a p53-dependent cell cycle arrest by canceling the inhibitory function of HDM2. Interference with ribosomal biogenesis by a low concentration of actinomycin D is associated with an increased L11-HDM2 interaction and subsequent p53 stabilization. We suggest that L11 functions as a negative regulator of HDM2 and that there might exist in vivo an L11-HDM2-p53 pathway for monitoring ribosomal integrity.     

Interplay between ribosomal protein S27a and MDM2 protein in p53 activation in response to ribosomal stress

Ribosomal proteins play a critical role in tightly coordinating p53 signaling with ribosomal biogenesis. Several ribosomal proteins have been shown to induce and activate p53 via inhibition of MDM2. Here, we report that S27a, a small subunit ribosomal protein synthesized as an 80-amino acid ubiquitin C-terminal extension protein (CEP80), functions as a novel regulator of the MDM2-p53 loop. S27a interacts with MDM2 at the central acidic domain of MDM2 and suppresses MDM2-mediated p53 ubiquitination, leading to p53 activation and cell cycle arrest. Knockdown of S27a significantly attenuates the p53 activation in cells in response to treatment with ribosomal stress-inducing agent actinomycin D or 5-fluorouracil. Interestingly, MDM2 in turn ubiquitinates S27a and promotes proteasomal degradation of S27a in response to actinomycin D treatment, thus forming a mutual-regulatory loop. Altogether, our results reveal that S27a plays a non-redundant role in mediating p53 activation in response to ribosomal stress via interplaying with MDM2.     

Aberrant expression of nucleostemin activates p53 and induces cell cycle arrest via inhibition of MDM2

The nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis. Both depletion and overexpression of NS reduce cell proliferation. However, the mechanisms underlying this regulation are still unclear. Here, we show that NS regulates p53 activity through the inhibition of MDM2. NS binds to the central acidic domain of MDM2 and inhibits MDM2-mediated p53 ubiquitylation and degradation. Consequently, ectopic overexpression of NS activates p53, induces G(1) cell cycle arrest, and inhibits cell proliferation. Interestingly, the knockdown of NS by small interfering RNA also activates p53 and induces G(1) arrest. These effects require the ribosomal proteins L5 and L11, since the depletion of NS enhanced their interactions with MDM2 and the knockdown of L5 or L11 abrogated the NS depletion-induced p53 activation and cell cycle arrest. These results suggest that a p53-dependent cell cycle checkpoint monitors changes of cellular NS levels via the impediment of MDM2 function.     

Ribosomal protein L23 activates p53 by inhibiting MDM2 function in response to ribosomal perturbation but not to translation inhibition

The p53-MDM2 feedback loop is vital for cell growth control and is subjected to multiple regulations in response to various stress signals. Here we report another regulator of this loop. Using an immunoaffinity method, we purified an MDM2-associated protein complex that contains the ribosomal protein L23. L23 interacted with MDM2, forming a complex independent of the 80S ribosome and polysome. The interaction of L23 with MDM2 was enhanced by treatment with actinomycin D but not by gamma-irradiation, leading to p53 activation. This activation was inhibited by small interfering RNA against L23. Ectopic expression of L23 reduced MDM2-mediated p53 ubiquitination and also induced p53 activity and G(1) arrest in p53-proficient U2OS cells but not in p53-deficient Saos-2 cells. These results reveal that L23 is another regulator of the p53-MDM2 feedback regulation.     

Ribosomal protein L11 recruits miR-24/miRISC to repress c-Myc expression in response to ribosomal stress

c-Myc promotes cell growth by enhancing ribosomal biogenesis and translation. Deregulated expression of c-Myc and aberrant ribosomal biogenesis and translation contribute to tumorigenesis. Thus, a fine coordination between c-Myc and ribosomal biogenesis is vital for normal cell homeostasis. Here, we show that ribosomal protein L11 regulates c-myc mRNA turnover. L11 binds to c-myc mRNA at its 3' untranslated region (3'-UTR), the core component of microRNA-induced silencing complex (miRISC) argonaute 2 (Ago2), as well as miR-24, leading to c-myc mRNA reduction. Knockdown of L11 drastically increases the levels and stability of c-myc mRNA. Ablation of Ago2 abrogated the L11-mediated reduction of c-myc mRNA, whereas knockdown of L11 rescued miR-24-mediated c-myc mRNA decay. Interestingly, treatment of cells with the ribosomal stress-inducing agent actinomycin D or 5-fluorouracil significantly decreased the c-myc mRNA levels in an L11- and Ago2-dependent manner. Both treatments enhanced the association of L11 with Ago2, miR-24, and c-myc mRNA. We further show that ribosome-free L11 binds to c-myc mRNA in the cytoplasm and that this binding is enhanced by actinomycin D treatment. Together, our results identify a novel regulatory paradigm wherein L11 plays a critical role in controlling c-myc mRNA turnover via recruiting miRISC in response to ribosomal stress.     

Mycophenolic acid activation of p53 requires ribosomal proteins L5 and L11

Mycophenolate mofetil (MMF), a prodrug of mycophenolic acid (MPA), is widely used as an immunosuppressive agent. MPA selectively inhibits inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme for the de novo synthesis of guanine nucleotides, leading to depletion of the guanine nucleotide pool. Its chemotherapeutic effects have been attributed to its ability to induce cell cycle arrest and apoptosis. MPA treatment has also been shown to induce and activate p53. However, the mechanism underlying the p53 activation pathway is still unclear. Here, we show that MPA treatment results in inhibition of pre-rRNA synthesis and disruption of the nucleolus. This treatment enhances the interaction of MDM2 with L5 and L11. Interestingly, knockdown of endogenous L5 or L11 markedly impairs the induction of p53 and G(1) cell cycle arrest induced by MPA. These results suggest that MPA may trigger a nucleolar stress that induces p53 activation via inhibition of MDM2 by ribosomal proteins L5 and L11.     

Depletion of ribosomal protein L37 occurs in response to DNA damage and activates p53 through the L11/MDM2 pathway

Perturbation of ribosomal biogenesis has recently emerged as a relevant p53-activating pathway. This pathway can be initiated by depletion of certain ribosomal proteins, which is followed by the binding and inhibition of MDM2 with a different subset of ribosomal proteins that includes L11. Here, we report that depletion of L37 leads to cell cycle arrest in a L11- and p53-dependent manner. DNA damage can initiate ribosomal stress, although little is known about the mechanisms involved. We have found that some genotoxic insults, namely, UV light and cisplatin, lead to proteasomal degradation of L37 in the nucleoplasm and to the ensuing L11-dependent stabilization of p53. Moreover, ectopic L37 overexpression can attenuate the DNA damage response mediated by p53. These results support the concept that DNA damage-induced proteasomal degradation of L37 can constitute a mechanistic link between DNA damage and the ribosomal stress pathway, and a relevant contributing signaling pathway for the activation of p53 in response to DNA damage.     

Nucleostemin: Another nucleolar “Twister” of the p53-MDM2 loop

Several nucleolar proteins, such as ARF, ribosomal protein (RP) L5, L11, L23 and S7, have been shown to induce p53 activation by inhibiting MDM2 E3 ligase activity and consequently to trigger cell cycle arrest and/or apoptosis. Our recent study revealed another nucleolar protein called nucleostemin (NS), a nucleolar GTP binding protein, as a novel regulator of the p53-MDM2 feedback loop. However, unlike other known nucleolar regulators of this loop, NS surprisingly plays a dual role, as both up and downregulations of its levels could turn on p53 activity. Here, we try to offer some prospective views for this unusual phenomenon by reconciling previously and recently published studies in the field in hoping to better depict the role of NS in linking the p53 pathway with ribosomal biogenesis during cell growth and proliferation as well as to propose NS as another potential molecular target for anti-cancer drug development.Key words: ribosomal biogenesis, nucleolar stress, nucleostemin, p53, MDM2, cell cycle, cell growth     

Loss of Ribosomal Protein L11 Affects Zebrafish Embryonic Development through a p53-Dependent Apoptotic Response

Ribosome is responsible for protein synthesis in all organisms and ribosomal proteins (RPs) play important roles in the formation of a functional ribosome. L11 was recently shown to regulate p53 activity through a direct binding with MDM2 and abrogating the MDM2-induced p53 degradation in response to ribosomal stress. However, the studies were performed in cell lines and the significance of this tumor suppressor function of L11 has yet to be explored in animal models. To investigate the effects of the deletion of L11 and its physiological relevance to p53 activity, we knocked down the rpl11 gene in zebrafish and analyzed the p53 response. Contrary to the cell line-based results, our data indicate that an L11 deficiency in a model organism activates the p53 pathway. The L11-deficient embryos (morphants) displayed developmental abnormalities primarily in the brain, leading to embryonic lethality within 6–7 days post fertilization. Extensive apoptosis was observed in the head region of the morphants, thus correlating the morphological defects with apparent cell death. A decrease in total abundance of genes involved in neural patterning of the brain was observed in the morphants, suggesting a reduction in neural progenitor cells. Upregulation of the genes involved in the p53 pathway were observed in the morphants. Simultaneous knockdown of the p53 gene rescued the developmental defects and apoptosis in the morphants. These results suggest that ribosomal dysfunction due to the loss of L11 activates a p53-dependent checkpoint response to prevent improper embryonic development.     

Ribosomal 18 S RNA Processing by the IGF-I-responsive WDR3 Protein Is Integrated with p53 Function in Cancer Cell Proliferation

Insulin-like growth factor-I (IGF-I) signaling is strongly associated with cell growth and regulates the rate of synthesis of the rRNA precursor, the first and the key stage of ribosome biogenesis. In a screen for mediators of IGF-I signaling in cancer, we recently identified several ribosome-related proteins, including NEP1 (nucleolar essential protein 1) and WDR3 (WD repeat 3), whose homologues in yeast function in ribosome processing. The WDR3 gene and its locus on chromosome 1p12-13 have previously been linked with malignancy. Here we show that IGF-I induces expression of WDR3 in transformed cells. WDR3 depletion causes defects in ribosome biogenesis by affecting 18 S rRNA processing and also causes a transient down-regulation of precursor rRNA levels with moderate repression of RNA polymerase I activity. Suppression of WDR3 in cells expressing functional p53 reduced proliferation and arrested cells in the G₁ phase of the cell cycle. This was associated with activation of p53 and sequestration of MDM2 by ribosomal protein L11. Cells lacking functional p53 did not undergo cell cycle arrest upon suppression of WDR3. Overall, the data indicate that WDR3 has an essential function in 40 S ribosomal subunit synthesis and in ribosomal stress signaling to p53-mediated regulation of cell cycle progression in cancer cells.     

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.     

Nucleostemin

Several nucleolar proteins, such as ARF, ribosomal protein (RP) L5, L11, L23 and S7, have been shown to induce p53 activation by inhibiting MDM2 E3 ligase activity and consequently to trigger cell cycle arrest and/or apoptosis. Our recent study revealed another nucleolar protein called nucleostemin (NS), a nucleolar GTP binding protein, as a novel regulator of the p53-MDM2 feedback loop. However, unlike other known nucleolar regulators of this loop, NS surprisingly plays a dual role, as both up and down regulations of its levels could turn on p53 activity. Here, we try to offer some prospective views for this unusual phenomenon by reconciling previously and recently published studies in the field in hoping to better depict the role of NS in linking the p53 pathway with ribosomal biogenesis during cell growth and proliferation as well as to propose NS as another potential molecular target for anti-cancer drug development.