The nucleolus as a key regulator of cellular metabolism and ribosome biogenesis in normal and transformed cells.

One of the central regulators of cell growth and adaptation to various stresses is mTOR kinase (mammalian target of rapamycin) (Liu and Sabatini, 2020; Ni and Buszczak, 2023). mTOR is a serine/threonine kinase from PI3K-related kinase family (Liu and Sabatini, 2020; Ni and Buszczak, 2023) which integrates both internal and external signals and coordinating diverse cellular processes, including cell growth, proliferation, and survival (Liu and Sabatini, 2020; Saba et al., 2021). It controls ribosome biogenesis and protein synthesis depending on external conditions and metabolic state of the cell. In addition, mTOR is involved in cellular responses to amino acid deprivation and oxidative stress, thus ensuring cell survival and function under changing conditions (Marini et al., 2018). mTOR forms two multiprotein complexes—mTORC1 and mTORC2 (Liu and Sabatini, 2020; Ni and Buszczak, 2023). mTORC1 mainly regulates cellular anabolism in response to nutrient and growth factor availability (Deleyto-Seldas and Efeyan, 2021). Downstream effectors of mTOR are the kinases S6K1 and S6K2 (Lai et al., 2010), which phosphorylate key substrates such as rpS6, IRS-1, and eIF4B, which promotes global translation and cell growth (Lai et al., 2010). mTORC1 also regulates the translation and function of multiple transcription factors such as hypoxia-inducible factor 1α (HIF1α) and c-MYC (Pourdehnad et al., 2013; Dodd et al., 2015; He et al., 2025).

mTORC1 signaling network affects various stages of ribosome biogenesis, including rRNA transcription, ribosomal protein synthesis, and assembly of functional ribosomes (Chauvin et al., 2014; He et al., 2025). mTORC1 promotes the translation of mRNAs containing a terminal oligopyrimidine (TOP) motif. The TOP motif is found in transcripts of all 79 human ribosomal protein genes, whose translation is tightly regulated and largely dependent on mTORC1 activity (Thoreen et al., 2012; Tao et al., 2020). mTOR and its effector kinase p70-S6 (S6K) control rRNA transcription by phosphorylating the initiation factor TIF-IA, a core component of the Pol I. Phosphorylation of TIFIA modulates both the nucleolar localization and activity of TIF-IA enhancing its interaction with Pol I and rate of rRNA synthesis (Mayer et al., 2004; Ni and Buszczak, 2023). Under nutrient-rich conditions, TIF-IA actively binds to Pol I and localizes to the nucleolus. However, inhibition of mTORC1 disrupts this interaction, accompanied by changes in TIF-IA phosphorylation (Mayer et al., 2004; Ni and Buszczak, 2023). In parallel, the mTORC1–S6K axis by phosphorylation regulates the activity UBF (Ni and Buszczak, 2023). mTOR-dependent activation increases UBF binding to SL1, another component of the Pol I pre-initiation complex, further enhancing rRNA transcription (Stefanovsky et al., 2006; Tao et al., 2020).

In addition to rRNA transcription, mTORC1 controls 5S rRNA synthesis by Pol III. First, mTORC1 phosphorylates and inactivates MAF1, main repressor of Pol III (Kantidakis et al., 2010; Michels et al., 2010; Shor et al., 2010; He et al., 2025). Second, mTORC1 directly promotes assembly of the Pol III complex by facilitating interactions between TFIIIB and TFIIIC (Mayer and Grummt, 2006). It was shown that mTORC1 may directly associate with TFIIIC, further enhancing 5S rRNA transcription (Kantidakis et al., 2010; Michels et al., 2010; Ni and Buszczak, 2023). Independent of effects on Pol I transcription, mTOR can modulate pre-rRNA processing (Iadevaia et al., 2014; Ni and Buszczak, 2023). By activating S6K1 and the ribosomal protein RPS6, mTORC1 enhances production of 40S ribosomal subunits thereby promoting cell growth (Jiao et al., 2023).

Notably, the mTORC2 complex component RICTOR and the enzyme L-glutamine synthetase (GLUL) have recently been identified in screens for human ribosome biogenesis factors (Badertscher et al., 2015; Bohnsack and Bohnsack, 2019). Since the impact of GLUL on 40S subunit biogenesis appears to be independent of mTORC1, intracellular glutamine synthesis may be essential for efficient ribosome production (Badertscher et al., 2015; Bohnsack and Bohnsack, 2019). mTOR also controls translation, enabling regulation of protein synthesis under stress conditions such as amino acid deficiency and oxidative stress. mTOR influences both global and selective mRNA translation (Thoreen et al., 2012; Marini et al., 2018). Taken together, these findings indicate that mTOR regulates ribosome biogenesis at multiple levels in response to fluctuating growth conditions.

Nucleolus, while being a site and factory for ribosome biogenesis, indirectly senses stress signals and initiates multiple signaling cascades to deal with environmental challenges (Boulon et al., 2010; Golomb et al., 2014). Of particular interest are signaling pathway involving ribosomal proteins RPL11 and RPL5, as well as 5S rRNA, which plays a unique role in transmitting stress signals upon disruption of ribosome biogenesis and activation of p53 activation (Bursac et al., 2014; Golomb et al., 2014).

p53 (encoded by TP53 gene) is a famous tumor suppressor responding to stresses such as DNA damage or impaired ribosome biogenesis by stabilization of its polypeptide, and hence, increasing concentration of active protein which level is remarkably low under normal conditions. p53 is a transcription factor controlling transcription of many genes, including p21/WAF1 (Figure 2), which is a negative regulator of the cell cycle. p21 inhibits G1phase cyclins preventing their binding to CDK (cyclin-dependent kinases) and arresting cells in G1 phase. In addition, p21 suppresses cyclin B, which is required for the G2/M transition. If DNA is damaged, p53 stimulates the synthesis of DNA repair proteins. The G1 arrest is further amplified through a positive feedback loop. DNA breaks increase level of p53 which activates its target genes (Vousden and Prives, 2009; Zilfou and Lowe, 2009; Bursac et al., 2014). Moreover, p53 inhibits both Pol I transcription by binding to the SL1, necessary for Pol I recruitment to the rDNA (Derenzini et al., 2017), and Pol III transcription by binding to TFIIIB (Felton-Edkins et al., 2003; Derenzini et al., 2017). Dysregulation of p53, observed in half of all human malignant tumors, leads to uncontrolled proliferation, genomic instability, and the evolution of stress-damaged cells, promoting their survival and malignant transformation (Levine and Oren, 2009; Zilfou and Lowe, 2009; Bursac et al., 2014). In tumor cells, where wild-type p53 is preserved, p53 functions are likely inactivated due to defects in upstream or downstream components of the p53 regulatory network (Vousden and Prives, 2009; Bursac et al., 2014).

The level of p53 is regulated post-translationally, ubiquitination of p53 is mediated by MDM2 and drives proteasomal degradation of p53 (Marchenko et al., 2007). Transcription of MDM2 gene is controlled by p53, forming a negative feedback loop releasing p53-mediated suppression of cell cycle after removal of the stress and repair of damage (Haupt et al., 1997). Stabilization of p53 requires cessation of its degradation by various means converging on the disruption of the p53–MDM2 interaction (Boyd et al., 2011). MDM2, an E3 ubiquitin ligase, possesses intrinsic enzymatic activity and can directly ubiquitinate p53. MDM4, a paralog of MDM2, lacks ubiquitin ligase activity but, by forming a heterodimer with MDM2, markedly enhances MDM2’s ability to ubiquitinate p53 (Figure 2). MDM2/MDM4 complexes suppress p53 activity and control its cellular levels using several mechanisms. By direct binding to p53, they block p53 ability to activate target genes. MDM2-mediated ubiquitination tags p53 for proteasomal degradation (Zhu et al., 2025) and regulates p53 transport. Mono-ubiquitination at low MDM2 levels promotes nuclear export of p53, which does not lead to degradation but is associated with mitochondrial translocation and apoptosis (Marchenko et al., 2007; Zhu et al., 2025). By contrast, high MDM2 levels promote polyubiquitination of p53, and its degradation in the 26S proteasome within the nucleus (Figure 2) (Boyd et al., 2011). Evidence suggests polyubiquitination of p53 occurs selectively within the nucleolus. This is supported by findings that mutations, preventing MDM2 nucleolar localization, specifically disrupt p53 polyubiquitination (Figure 2) (Vlatković et al., 2014). Both p53 and MDM2 continuously and rapidly shuttle through the nucleolus, such that nearly all p53 molecules (or at least the vast majority) reside there at some point. Nucleolar p53 is ubiquitinated with a characteristic pattern showing more polyubiquitination than in the nucleoplasm or cytoplasm (Boyd et al., 2011). Upon proteasome inhibition, polyubiquitinated p53 accumulates in nucleoli (Vlatković et al., 2014). Non-mutually exclusive models have been proposed to explain how nucleolar disruption stabilizes p53. In the relocalization model, stress triggers the relocalization of nucleolar proteins (e.g., ribosomal protein L11) into the nucleoplasm, where they bind MDM2 and inhibit its ability to ubiquitinate p53. In the direct nucleolar control model, stress directly impairs efficient polyubiquitination of p53, which normally occurs in the nucleolus, even in the absence of detectable protein relocalization (Vlatković et al., 2014). Importantly, although MDM2 is the predominant E3 ligase for p53 in vivo, about twenty p53-targeting ubiquitin E3 ligases have been identified to date. Some of these restrict nuclear accumulation of p53, while others facilitate its nuclear export, thereby promoting cytoplasmic retention (Rodríguez, 2014). The ability of the tumor suppressor p53 to form dimers and tetramers represents another regulatory layer of its activity, stability, and subcellular localization. In the absence of stress, p53 exists predominantly as dimers (∼59%) and monomers (∼28%), with tetramers—required for its function—being relatively scarce (∼13%) (Holoubek et al., 2025). p53 oligomerization status can modulate cell fate decisions between growth, cell cycle arrest and apoptosis (Fischer et al., 2016). Upon activation, for example after DNA damage, the proportion of functional tetramers increases sharply, reaching ∼93% of total p53. This occurs both due to overall p53 accumulation and the action of additional factors that accelerate tetramer assembly. In some stress contexts, an increase in tetramer content precedes the rise in overall p53 levels. Certain post-translational modifications, such as phosphorylation or acetylation, accelerate tetramerization. The tetrameric form of p53 binds DNA ∼1,000 times more efficiently than the monomer, making it a potent transcription factor. Some critical post-translational modifications, such as phosphorylation at Ser15—which impairs MDM2 binding—occur only after tetramerization. p53 possesses a nuclear export signal (NES) (Figure 2) (Holoubek et al., 2025). In monomers and dimers, this signal is exposed, enabling export to cytoplasm. In tetramers, the NES is masked, retaining active p53 in the nucleus. MDM2-mediated ubiquitination of p53 also depends on its oligomeric state. Tetramers are efficiently ubiquitinated by MDM2 and degraded via the ubiquitin-dependent 26S proteasome pathway (Figure 2). By contrast, monomers and dimers (including oligomerization-domain mutants) are poorly ubiquitinated by MDM2, but can be degraded by an alternative, ubiquitin-independent proteasomal pathway (Gencel-Augusto and Lozano, 2020). For example, MYBBP1A, a protein involved in rRNA transcription and processing, modulates p53 oligomerization. In the presence of ectopically expressed MYBBP1A, p53 is predominantly detected in high-molecular-weight fractions (Ono et al., 2014; Gencel-Augusto and Lozano, 2020). Interestingly, p53 dimers bind MDM2 more efficiently than monomers and undergo ubiquitin-independent degradation. It was found (Kulikov et al., 2010) that MDM2 can directly bind the proteasome, enhancing p53–proteasome association and promoting efficient degradation; however, whether MDM2 plays an active role in modulating p53 oligomerization remains an open question (Gencel-Augusto and Lozano, 2020).

It has been proposed that translocation of the p53/MDM2 complex to the cytoplasmis required for their proteasomal degradation although there are evidence that proteasome-mediated p53 degradation can also occur in the nucleus (Shirangi et al., 2002; Golomb et al., 2014). Moreover, it likely can occur even in the nucleolus in a ubiquitin-independent, calpain-dependent manner (Tao et al., 2013; Golomb et al., 2014). Activity of p53 is also dependent on post-translational modifications such as phosphorylation (Golomb et al., 2014) and acetylation (Brooks and Gu, 2011; Golomb et al., 2014) of p53 or MDM2 (Golomb et al., 2014). These modifications disrupt p53/MDM2 interaction, promote p53 tetramerization, and enhance its binding to target genes (Golomb et al., 2014). NPM1 facilitates MDM2 dephosphorylation, thereby enhancing p53 degradation (Maggi et al., 2014; Luchinat et al., 2018; González-Arzola et al., 2022). Furthermore, NPM1 directly interacts with p53, suppressing tumor cell growth, and this interaction activates p53-mediated transcription (Luchinat et al., 2018). Several observations suggested existence of a cell cycle checkpoint triggered by disruption of ribosome biogenesis (Bursac et al., 2014; Dong et al., 2020). Expression of dominant-negative mutants of Bop1 (a pre-60S biogenesis factor and member of the PeBoW complex) and several other factors inhibiting rRNA processing has been shown to prevent cell cycle progression in a p53-dependent manner. Similarly, inhibition of rRNA processing by 5-fluorouracil (Sun et al., 2007; Nishimura et al., 2015) or reduced expression of proteins required for 18S and 28S rRNA maturation—such as hUTP1 (Hölzel et al., 2005), PAK1IP1(Yu et al., 2010), WDR3 (Yu et al., 2010), WDR12 (Hölzel et al., 2005), WDR36 (Skarie and Link, 2008), nucleophosmin (NPM, B23) (Skarie and Link, 2008), nucleostemin (Table 1) (Dai et al., 2008), and multiple 40S or 60S ribosomal proteins (Table 2) (Fumagalli et al., 2009) can also induce p53-mediated stress signaling. Importantly, for most ribosome biogenesis factors, pre-rRNA processing defects caused by their depletion were p53-independent. For example, in (Tafforeau et al., 2013), pre-rRNA processing defects were examined after depletion of 21 factors in HCT116 cells in the presence or absence of p53, and the resulting phenotypes were nearly identical. However, for some factors, such as SURF6, processing defects after knockdown were p53-dependent and did not trigger p53-mediated cell cycle arrest (Moraleva et al., 2023).

Subsequent studies showed that other disruptions in ribosome biogenesis can also trigger a p53 response (Zhang and Lu, 2009; Bursac et al., 2014). Inhibition of rRNA transcription can lead to nucleolar functional changes and p53 stabilization (Bywater et al., 2012; Bursac et al., 2014). Additionally, blocking nuclear import or export of ribosomal subunits disrupts ribosome biogenesis and activates p53 response (Daftuar et al., 2013; Bursac et al., 2014). Taken together, these findings suggest that inhibiting ribosome biogenesis at various levels often leads to subsequent p53 accumulation.

Ribosomal proteins RPL5 and RPL11 are directly involved in activation of p53. Early studies showed that RPL5 forms an extraribosomal complex with MDM2, p53, and 5S rRNA (Marechal et al., 1994; Bursac et al., 2014). Later works demonstrated that R,PL5 and RPL11 bind MDM2, blocking its E3 ligase function and promoting p53 accumulation (Dai et al., 2004). Depletion of RPL5 or RPL11is sufficient to suppress p53 activation upon ribosome biogenesis inhibition at various levels, as these proteins are interdependently essential for this response (Bursać et al., 2012; Fumagalli et al., 2012; Zhou et al., 2012b; Bursac et al., 2014).

The interaction of RPL5 and RPL11 with MDM2 is not unique, as ribosomal proteins—such as RPL23 (Nishimura et al., 2015; Turi et al., 2019), RPL26 (Ofir-Rosenfeld et al., 2008; Zhang et al., 2010; Micic et al., 2020) and others (Table 2), when overexpressed in cells bind MDM2 and inhibit its ubiquitin ligase activity thereby upregulating p53 (Warner, 1977; Bursac et al., 2014).

Interestingly, depletion of RPL5 or RPL11 inhibits ribosome biogenesis (Fumagalli et al., 2012; Donati et al., 2013; Bursac et al., 2014) but does not trigger a p53 response. This suggests that RPL5 and RPL11 are important transducers of p53 activation signals during ribosome biogenesis stress (Zhang and Lu, 2009; Deisenroth and Zhang, 2010; Bursać et al., 2012; Fumagalli et al., 2012; Bursac et al., 2014). In contrast, ribosome biogenesis inhibition caused by depletion of RPL23, RPL26, or RPS7— as well as depletion of other ribosomal proteins except RPL11 and RPL5—activates p53 in an RPL5/RPL11-dependent manner (Bursać et al., 2012; Fumagalli et al., 2012; Bursac et al., 2014). It has been proposed that depletion of specific ribosomal proteins may reduce ribosome numbers, and their inhibitory effect on p53 accumulation may be explained by global translational suppression rather than loss of their specific effects on MDM2 function (Fumagalli et al., 2012; Bursac et al., 2014). Alternative hypothesis suggests that a number of ribosomal proteins, including RPL5, RPL11, RPL23, RPL26, and RPS7, passively diffuse from the nucleolus into the nucleoplasm, where they bind MDM2 and inhibit its anti-p53 activity (Deisenroth and Zhang, 2010; Bursać et al., 2012; Zhou et al., 2012a; Bursac et al., 2014). However, nucleolar disruption and passive diffusion of ribosomal proteins do not necessarily occur during ribosome stress; but may result in increased translation of RPL5 and RPL11 mRNA.

Excessive production and nuclear import of ribosomal proteins may be sufficient for p53 activation (Donati et al., 2011; Bursac et al., 2014). In particular, impaired 40S ribosome biogenesis increases RPL11 mRNA transcription and does not significantly affects 60S biogenesis or nucleolar integrity (Nishimura et al., 2015). Excessive RPL11 presumably translocates to the nucleus, where it interacts with MDM2, and blocks its function, resulting in p53 stabilization. In contrast, inhibition of 60S biogenesis impairs RPL11 mRNA translation (Nishimura et al., 2015; Zhou et al., 2015). Most ribosomal proteins are synthesized during ribosome biogenesis inhibition but undergo ubiquitin-independent proteasomal degradation to prevent toxic accumulation of free ribosomal proteins (Andersen et al., 2005; Bursać et al., 2012; Bursac et al., 2014). Conversely, RPL5, RPL11, and 5S rRNA are redirected from 60S ribosome assembly to MDM2 inhibition in the cytoplasm and nucleoplasm during ribosome stress (Bursać et al., 2012; Donati et al., 2013; Bursac et al., 2014). An additional complication is the existence of a homologue of MDM2, namely MDMX (also known as HDMX/MDM4/HDM4), which works in concert with MDM2 to degrade p53 (Klein et al., 2021). Thus, 5S rRNA may act as a positive or negative regulator of p53, depending on its association with the RPL5-RPL11-MDM2 complex or MDMX, respectively. In this context, it is possible that the L11-MDM2-p53 signaling pathway may mediate a p53-dependent checkpoint in response to deregulated oncogenes that promote excessive ribosome biogenesis (Macias et al., 2010).

Interaction between MDM2 and p53 could be prevented by binding of modulator proteins to MDM2, directly or by sequestering MDM2 in a different cellular compartment (Weber et al., 1999; Golomb et al., 2014). The best-known example of this modulators is the tumor suppressor ARF (p14ARF in humans, p19ARF in mice). This protein accumulates under excessive mitogenic signaling, such as c-MYC overexpression, and induces p53-dependent apoptosis or growth arrest (Lessard et al., 2010; Golomb et al., 2014). The primary mechanism of ARF action is MDM2 inhibition, which prevents p53 degradation and enhances its antitumor activity (Kung and Weber, 2022) in response to a broad range of oncogenic stimuli, including increased expression of MYC, E2F1, RAS, E1A, and v-Abl (Macias et al., 2010). Mutations in ARF are found in about 40% of tumors (Lessard et al., 2010). Transgenic mice lacking ARF spontaneously developed tumors over 1 year, which include sarcomas, lymphomas, carcinomas, and nervous system tumors (Lahav et al., 2004; Kung and Weber, 2022). It has been shown that ARF can inhibit the cell cycle even in the absence of p53 mediating p53-independent tumor suppression (Figures 3, 4) (Rocha et al., 2003; Kelly-Spratt et al., 2004; Sandoval et al., 2004; Korgaonkar et al., 2005; Muniz et al., 2011; Forys et al., 2014; Kung and Weber, 2022). ARF is encoded by the CDKN2A locus, which also encodes another tumor suppressor, p16INK4A. ARF and p16INK4A are transcribed from two partially overlapping open reading frames and translated into two unrelated proteins. Two arginine-rich domains (amino acids 1–14 and 82–101) of ARF mediate its nucleolar localization (Kung and Weber, 2022). The N-terminal motif (amino acids 1–14) of ARF interacts with the central domain of MDM2 inhibiting its ubiquitin ligase activity (Figures 3, 4) (Lohrum et al., 2000; Weber et al., 2000; Kung and Weber, 2022). ARF inhibits MDM2 by preventing its translocation from the nucleoplasm to the nucleolus (Figure 3) (Lin and Lowe, 2001; Llanos et al., 2001; Korgaonkar et al., 2005). Notably, even non-nucleolar forms of ARF can activate p53 and suppress proliferation (Korgaonkar et al., 2005), indicating that nucleolar localization of ARF is not essential for its function. Besides MDM2, ARF binds numerous other proteins, sequestering them in the nucleolus upon overexpression (Korgaonkar et al., 2005). The interaction between ARF and NPM1 in the nucleolus increases ARF stability and functional activity (Figures 3, 4) (Pandit and Gartel, 2011; Maggi et al., 2014; Luchinat et al., 2018). NPM1 protects ARF from proteasomal degradation by retaining it in the nucleolus, and also controls ARF availability (Quin et al., 2014; González-Arzola et al., 2022; Riback et al., 2023). Most cellular ARF appears bound to NPM1, whereas only a small fraction of NPM1 associates with ARF. Under normal conditions, this interaction maintains a balance allowing ARF to persist in cells and remain inactive. Upon cellular stress such as DNA damage or oncogenic activation, ARF is released from the NPM1 complex (Lee et al., 2005; González-Arzola et al., 2022), enabling its exit to the nucleoplasm interaction with MDM2, blockage of MDM2 ubiquitin ligase activity and stabilization of p53 (Figures 3, 4) (Rubbi and Milner, 2003). Knockdown of NPM1 specifically reduces nucleolar localization of ARF, significantly enhances the association of ARF with human MDM2, and activates p53, while NPM1 overexpression counteracts ARF function by increasing ARF nucleolar retention. In acute myeloid leukemia associated with NPM1 mutations, this mechanism is disrupted. Overexpression or mutant forms of NPM1 causes abnormal sequestration of ARF in the nucleolus, preventing its interaction with MDM2 and subsequent p53 activation (Colombo et al., 2006; Moulin et al., 2008; Kung and Weber, 2022). Interestingly, the ARF-NPM1 relationship constitutes a complex negative feedback system (Figure 4). While NPM1 regulates ARF localization and stability, ARF itself can affect NPM1 expression by reducing its stability and function, thereby impairing its role in rRNA processing (Korgaonkar et al., 2005; González-Arzola et al., 2022; Kung and Weber, 2022). This mutual regulation creates a delicate balance controlling proliferation of normal cells and contributing to tumor growth, when disrupted. Notably, even under physiological conditions, such as in aging cells, most ARF remains in complex with NPM1, highlighting the importance of this interaction for cellular homeostasis (Bertwistle et al., 2004; Korgaonkar et al., 2005). The ARF-NPM interaction is also sensitive to stress factors, including AKT, cytochrome c, and CD24 (Brady et al., 2004; Hamilton et al., 2014; Kung and Weber, 2022). As a nucleolar protein, ARF suppresses tumor growth by disrupting ribosome biogenesis, rRNA processing (Sugimoto et al., 2003; Lessard et al., 2010), and its translation (Cottrell et al., 2020; Kung and Weber, 2022). ARF regulates these processes through multiple mechanisms: direct interaction with the rRNA promoter, hindrance of RNA polymerase I transcription termination factor (TTF-I) import into the nucleolus (Colombo et al., 2005; Lessard et al., 2010), inactivation of UBF (Ayrault et al., 2006), suppression of the rRNA processing enzyme Drosha, and modulation of nucleolar localization of the RNA helicase DDX5 (Saporita et al., 2011; Kuchenreuther and Weber, 2014; Kung and Weber, 2022). Notably, ARF’s ability to interact with DDX5 also prevents DDX5-c-MYC interaction, disrupting a positive feedback loop that enhances c-MYC-mediated transcription (Sugimoto et al., 2003; Kung and Weber, 2022). Among many regulators, mTOR (mechanistic target of rapamycin) and the oncogene c-MYC affect ARF, MDM2 and p53 levels and activity, controlling malignant transformation processes (Figure 4) (Maggi et al., 2014; Hafner et al., 2019; Klein et al., 2021; Kung and Weber, 2022) (Figures 3, 4). Under cellular stress, p53 suppresses mTOR activity in two ways: via activation of the TSC1/TSC2 complex (mTOR inhibitor) involving AMPK kinase and sestrin-1/2 proteins, or by stimulating PTEN synthesis, which blocks AKT—an mTOR activator (Feng et al., 2005; Budanov and Karin, 2008; Kung and Weber, 2022). Conversely, oncogenic activation enhances mTOR-mediated translation of ARF mRNA boosting p53 activity and suppressing tumor growth. Additionally, mTOR can activate p53 through the kinase S6K1, which phosphorylates MDM2, impairing its nucleolar localization and thus stabilizing p53 (Lee et al., 2007; Lai et al., 2010; Kung and Weber, 2022). Therefore, mTOR and p53 mutually regulate each other, playing a key role in tumor suppression. c-MYC induces ARF expression and p53-dependent apoptotic programs in the initial DNA damage response but ultimately leads to inactivation of the ARF-MDM2-p53 pathway (Nieminen et al., 2013; Phesse et al., 2014; Kung and Weber, 2022). To suppress c-MYC-driven tumorigenesis, p53 can transcriptionally repress c-MYC directly by promoting histone deacetylation or indirectly via induction of microRNA (miR)-145 (Ho et al., 2005; Sachdeva et al., 2009; Kung and Weber, 2022). ARF directly interacts with c-MYC or its transcriptional cofactor Miz1 to induce growth arrest and cell death even in the absence of p53 (Herkert et al., 2010; Kung and Weber, 2022). Two parallel MDM2-mediated pathways maintain p53 activity to counteract oncogenic functions of c-MYC. Besides ARF-MDM2 interaction, ribosomal proteins interacting with MDM2 are also required to maximize p53 activity in inhibiting c-MYC-driven tumorigenesis (Macias et al., 2010; Kung and Weber, 2022). Recently, c-MYC was shown to regulate the p53-MDM2-ARF tumor suppression axis by modulating two different long noncoding RNAs (Jain, 2020; Xu et al., 2020; Kung and Weber, 2022). One, SENEBLOC, acts as a scaffold promoting p53-MDM2 association leading to p53 degradation. The other, MILIP, inhibits SUMOylation, reducing p53 SUMO modification (Kung and Weber, 2022). SUMOylation of p53 is an important post-translational modification mechanism by which MDM2 and ARF regulate p53 functions (Kung and Weber, 2022). ARF also mediates SUMOylation of NPM and MDM2 (Tago et al., 2005; Kung and Weber, 2022). c-MYC expression strongly elevates p53 level in ARF-null MEF cells. Given c-MYC’s role in stimulating ribosome biogenesis, this ARF-independent induction of p53 by c-MYC is likely mediated by ribosomal proteins. This suggests that p53 protects against c-MYC oncogenesis through two independent signaling pathways: ARF-MDM2-p53 and RP-MDM2-p53. Accordingly, disruption of either accelerates c-MYC-induced tumorigenesis (Macias et al., 2010). Data confirm the RPL11-MDM2-p53 pathway is an important in vivo barrier against c-MYC-driven oncogenesis, alongside ARF-MDM2-p53 signaling. Together, these results highlight the complexity of ribosome biogenesis regulation via the p53-ARF axis, suggesting this tumor suppressive pathway is a crucial barrier to tumorigenesis.

The study of MDM inhibitors has become a promising direction in cancer therapy, with numerous candidates reaching clinical trials. Nutlin-3a and RG7388 (Idasanutlin) are MDM2-p53 interaction inhibitors. Unlike direct Pol I inhibitors, Nutlin-3a and RG7388 aim to restore p53 function in tumors carrying wild-type TP53. They bind to the p53-binding pocket of the MDM2, preventing p53 degradation and leading to its stabilization and activation (Zhu et al., 2025). Nutlin-3a was the first compound, demonstrating preclinical efficacy, but its clinical application is hindered by low bioavailability, rapid systemic clearance, and the development of resistance, often associated with acquired TP53 mutations (Zhu et al., 2025). RG7388 is next-generation compound with nanomolar activity and increased selectivity. It showed promising results in early clinical trials. However, a large Phase III trial (MIRROS) evaluating its combination with cytarabine for acute myeloid leukemia did not show an improvement in overall survival compared to cytarabine alone. Furthermore, treatment was often accompanied by significant gastrointestinal and hematological toxicities, highlighting challenges with the therapeutic window (Zhu et al., 2025).

The existence of a p53-independent pathway regulating the interconnection between ribosome biogenesis and cell proliferation in mammals was first postulated based on the observation that selective inhibition of rRNA synthesis by actinomycin D could disrupt cell cycle progression even in p53-depleted cells, although to a lesser extent than in p53-proficient cells (Donati et al., 2012; Maehama et al., 2023). An example is the depletion of the Pes1 protein. Knockdown of Pes1—a nucleolar protein that is part of the PeBoW complex with Bop1 and WDR12 and is involved in pre-60S ribosome biogenesis—caused defects in ribosome biogenesis, stabilized p53, and led to cell cycle arrest (Grimm et al., 2006; Donati et al., 2012). However, Pes1 knockdown also impaired proliferation in p53-deficient cells, indicating the presence of p53-independent cell cycle control in these cells (Li et al., 2009; Donati et al., 2012). The underlying regulatory mechanism was associated with downregulation of the cell cycle protein cyclin D1 and upregulation of the CDK inhibitor p27, which plays a critical role in controlling the G1/S transition, leading to significant cell cycle slowdown (Montanaro et al., 2007; Morishita et al., 2008; Iadevaia et al., 2010).

One mechanism for stabilizing p27 in p53-deficient cells involves nucleolar stress-induced destabilization of the proto-oncogenic serine/threonine kinase PIM1 (Iadevaia et al., 2010; Olausson et al., 2012). Under normal conditions, PIM1 phosphorylates p27, targeting it for subsequent degradation via the ubiquitin-proteasome pathway. Disruption of ribosome biogenesis leads to a reduction in the level or activity of PIM1 kinase. The decrease in PIM1-mediated p27 phosphorylation, in turn, prevents its proteasomal degradation, resulting in the accumulation of stable p27 protein in the cell (Iadevaia et al., 2010; González-Arzola, 2024).

Other key players in p53-independent pathways are E2F-1, MYC, NFκB, and ribosomal proteins (Maehama et al., 2023). E2F-1 is responsible for activating the transcription of a broad spectrum of genes necessary for the G1/S transition of the cell cycle (Pfister et al., 2015; Pecoraro et al., 2019). A central mechanism for regulating E2F activity is its binding to the retinoblastoma protein (pRB), which occurs in a cell cycle-dependent manner. It has been shown that NPM1 participates in transporting the hyperphosphorylated form of pRB into the nucleolus. This process facilitates the dissociation of the pRB-E2F complex, releasing the active E2F factor, which then initiates the transcription of genes required for DNA synthesis (Takemura et al., 2002; Taha and Ahmadian, 2024). Under normal conditions, the stability of one of the key members of this family, E2F-1, is maintained through its interaction with the ubiquitin ligase MDM2. This interaction protects E2F-1 from proteasomal degradation by preventing the binding of other E3 ubiquitin ligases responsible for its polyubiquitination (Iadevaia et al., 2010; Donati et al., 2012). Upon disruption of ribosome biogenesis, the ribosomal protein RPL11 is released and migrates from the nucleolus to the nucleoplasm. RPL11 specifically binds to MDM2, blocking its stabilizing function towards E2F-1 (Donati et al., 2011; 2012). This, in turn, promotes MDM2-mediated polyubiquitination of E2F-1 and its subsequent degradation by the proteasome. Importantly, unlike the classical p53-dependent stress response where RPL11 binding to MDM2 stabilizes p53, in the case of E2F-1, the same interaction accelerates its degradation (Maehama et al., 2023).

RPL11, along with other ribosomal proteins such as RPL5 and RPS14, is involved in p53-independent cell cycle arrest, directly interacting with c-MYC mRNA by binding to its 3′-UTR and suppressing translational (Challagundla et al., 2011; Donati et al., 2012; Maehama et al., 2023). Concurrently, RPL11 can bind to the c-MYC protein itself, inhibiting its transcriptional activity (Dai et al., 2007; Olausson et al., 2012). It is important to emphasize that this suppression of c-MYC activity is also observed in cells lacking functional p53, defining it as a p53-independent mechanism for controlling proliferation (Dai et al., 2007).

RPL3, large ribosomal subunit protein, also mediates p53-independent pathways. Like other ribosomal proteins, RPL3 translocates from the nucleolus to the nucleoplasm in response to stress, including genotoxic stress (Pecoraro et al., 2019; González-Arzola, 2024). A central target of RPL3’s extraribosomal function is the potent CDK inhibitor p21 (CDKN1A). RPL3 activates p21 expression at multiple levels by directly binding to transcription factor Sp1 and increasing transcription of the CDKN1A gene (Maehama et al., 2023), and by stabilizing the p21 protein (Maehama et al., 2023). This combination of effects results in p21-dependent cell cycle arrest in p53-null cells in response to disruption of ribosome biogenesis (Russo et al., 2013; González-Arzola, 2024). In nucleoplasm, RPL3 binds to poly-ADP-ribose polymerase 1 (PARP-1). Preventing PARP-1 binding to the E2F1 gene promoter, thereby blocking its transcription (Pecoraro et al., 2019; González-Arzola, 2024). Furthermore, RPL3 negatively regulates cyclin D1 by reducing its mRNA and protein levels (Pecoraro et al., 2019; González-Arzola, 2024). The biological significance of the RPL3-mediated pathway is confirmed by its role in therapy response. Notably, RPL3 expression is decreased in colorectal cancer cells, which is associated with reduced efficacy of some chemotherapeutic agents (González-Arzola, 2024). Conversely, RPL3 enhances the activity of antitumor drugs, exhibiting pro-apoptotic and anti-autophagic effects, especially in p53-deficient cancer cells (Pecoraro et al., 2020; González-Arzola, 2024). These data highlight the potential of the RPL3 pathway as a target for therapeutic modulation.

Some ribosome biogenesis factors can regulate the cell cycle through both p53-dependent and p53-independent pathways. For example, the SURF6–Rrp15–PPAN (Table 1) complex is involved in pre-60S ribosome maturation and cell cycle regulation (Broeck and Klinge, 2023; Vanden Broeck and Klinge, 2024). Depletion of Rrp15 in human and mouse cells causes arrest at the G1/S checkpoint and increases p21 expression (Wu et al., 2016). In p53-deficient cells, Rrp15 knockdown induced a metabolic shift from glycolysis to mitochondrial oxidative phosphorylation and the accumulation of reactive oxygen species (ROS) triggering cell death. Similarly, depletion of the PPAN protein inhibited apoptosis in a p53-independent manner by stabilizing BAX and inducing mitochondrial depolarization (Pfister et al., 2015).

Thus, accumulating data demonstrate that ribosome stress impedes the proliferation of mammalian cells both in the presence and absence of p53. Given that many cancers are characterized by the absence of functional p53, understanding these p53-independent pathways is of critical importance (James et al., 2014; D’Orazi, 2023). p53 stabilization in response to ribosome stress plays a key role in preventing neoplastic transformation (Macias et al., 2010; Donati et al., 2012). Disrupted ribosome biogenesis may underlie defects in p53 activation pathways and contribute to tumor development (Montanaro et al., 2012).

It is well known that enhanced translational capacity resulting from increased ribosome biogenesis promotes cancer development and progression (Johnson et al., 1976; Bilanges and Stokoe, 2007; White, 2008; Bursac et al., 2014). Evidence suggests that the RPL5-RPL11-MDM2-p53 pathway may regulate excessive ribosome biogenesis to prevent oncogenesis (Oskarsson and Trumpp, 2005; Van Riggelen et al., 2010; Golomb et al., 2012; Destefanis et al., 2020). Independent signaling pathways of RPL5-RPL11-MDM2-p53 and ARF-MDM2-p53 control excessive ribosome biogenesis to protect cells from MYC-induced oncogenesis (Ruggero, 2009; Macias et al., 2010; Van Riggelen et al., 2010; Destefanis et al., 2020). At the same time, RPL5-RPL11 may play a role in oncogenesis independent of the MDM2–p53 module. The ARF gene can be mutated or silenced in tumor cells (Lowe and Sherr, 2003; Montanaro et al., 2012; Maggi et al., 2014), and loss of ARF expression may be responsible for enhanced ribosome biogenesis both directly and through effects on p53 stabilization (Montanaro et al., 2012).

Increased expression of ribosomal proteins is also associated with increased proliferation and growth (Van Riggelen et al., 2010). There have been numerous instances of ribosomal protein overexpression across many different cancer types. For example, RPLP0, RPLP1, and RPLP2 levels are found to be upregulated at the RNA level in patients with gynecologic tumors, and this upregulation positively correlates with the presence of lymph node metastases in ovarian cancer (Artero-Castro et al., 2011; Weeks et al., 2019). Furthermore, the overexpression of ribosomal proteins RPS11 and RPS20, both collectively and individually, has been found to be a predictor of poor survival outcomes in patients with glioblastoma (Yong et al., 2015; Weeks et al., 2019). In colorectal cancer cell lines HCT116 and HT-29, knockdown of RPS24 was found to inhibit cell migration and proliferation (Wang et al., 2015; Weeks et al., 2019). Conversely, overexpression of RPL19 in the breast cancer cell line MCF7 rendered the cells more susceptible to stress-induced cell death (Hong et al., 2014; Weeks et al., 2019).

Although many reports identify mutations in ribosomal proteins (RPs) occurring in cancer, it is hard to prove whether these mutations are oncogenic factors. These mutations are often reported alongside mutations in tumor suppressor genes and are considered as secondary due to genomic instability. However, several studies suggest that disregulated RPs could possibly induce oncogenesis. For example, RPL5, RPL14, and RPL22 are negative regulators of c-MYC (Rao et al., 2012; Liao J. M. et al., 2014; Zhou et al., 2015). Therefore, mutations in these RPs cause c-MYC overexpression and may contribute to transformation (Rao et al., 2012; Morgado-Palacin et al., 2015). New data indicate that RPs also regulate the NF-κB family of transcription factors. Characterization of approximately 500 lines of Danio rerio with heterozygous mutations in recessive lethal genes for early lethality and tumor development identified 12 lines with increased cancer incidence, 11 of which carry heterozygous mutations in RP genes (Zhang and Lu, 2009). Accordingly, aberrant RP expression in mouse models can stimulate oncogenesis (Rao et al., 2012). In humans, hyperplastic and dysplastic changes in epithelial cells promote neoplastic transformation (Montanaro et al., 2012). Human tissues and organs chronically affected by inflammatory diseases display increased tumor incidence. However, a common feature of neoplastic transformation prone conditions is nucleolar hypertrophy (Derenzini et al., 2009; Montanaro et al., 2012; Jia et al., 2024). This indicates that the rate of ribosome biogenesis is increased in hyperplastic and dysplastic cells, as nucleolar size directly correlates with Pol I transcriptional activity (Derenzini et al., 1998; Montanaro et al., 2012). The upregulation of ribosome biogenesis appears to be a consequence of high metabolic demands induced by hyperproliferation. Recent data indicate that increased ribosome biogenesis rates can indeed promote cell progression toward neoplastic transformation (Montanaro et al., 2012; Penzo et al., 2019). Qualitative alterations in ribosome biogenesis can be a reason for development of human diseases, primarily a group of rare inherited disorders called ribosomopathies (Narla and Ebert, 2010). In these diseases, genes encoding factors required for ribosome production—such as ribosomal proteins or other factors involved in rRNA transcription and processing—are mutated. These disorders include congenital X-linked dyskeratosis congenita (X-DC), Shwachman-Diamond syndrome (SDS), cartilage-hair hypoplasia (CHH), Diamond-Blackfan anemia (DBA), and Treacher Collins syndrome (Narla and Ebert, 2010; Golomb et al., 2014). DBA is characterized by bone marrow failure due to inhibited differentiation of hematopoietic stem cells along the erythroid lineage (Narla and Ebert, 2010; Golomb et al., 2014). Mutations associated with DBA have been identified in eleven different genes encoding both small and large subunit RP (Boria et al., 2010; Bohnsack and Bohnsack, 2019). Ribosomopathies have diverse phenotypes, but many share common or overlapping features such as hematopoietic dysfunction (e.g., DBA, 5q syndrome, congenital dyskeratosis, Shwachman-Diamond syndrome) and/or skeletal defects (especially craniofacial) (e.g., Treacher Collins syndrome, Bowen-Conradi syndrome, and RPS23-related ribosomopathy). Understanding how defects in a fundamental and ubiquitous cellular process like ribosome synthesis cause tissue-specific human pathologies remains a major challenge, and several models have been proposed to explain these phenomena (Mills and Green, 2017; Bohnsack and Bohnsack, 2019; Emmott et al., 2019). Since mutations in genes encoding RPs or ribosome biogenesis factors disrupt ribosome assembly, they lead to p53 stabilization via the 5S RNP/MDM2 pathway described above. Indeed, many tissue-specific defects observed in ribosomopathies appear to be p53-dependent, and symptoms of ribosomopathies such as Treacher Collins syndrome and 5q syndrome can be alleviated by inhibiting p53 function (Bohnsack and Bohnsack, 2019; Emmott et al., 2019). It has been suggested that differences in p53 activation threshold or degree among cell types underlie tissue specificity in many ribosomopathies. An alternative hypothesis posits that differences in ribosome composition among cell types and/or tissues lead to specialized translation. Mutations that reduce specific RP levels or disrupt rRNA modification mechanisms may thereby reduce or alter the production of proteins particularly required in certain cell types. Another possible molecular basis for tissue-specific effects in ribosomopathies could be translational changes caused by high ribosome demand in rapidly proliferating tissues. Supporting this, DBA-associated mutations reduce ribosome levels in hematopoietic cells, so ribosome availability becomes limiting, affecting translation of a subset of mRNAs, which in turn impairs erythroid lineage commitment (Khajuria et al., 2018; Bohnsack and Bohnsack, 2019). It has been proposed that differential effects on mRNA translation caused by ribosome scarcity arise from specific mRNA features (e.g., highly structured 5′-UTRs), rendering these transcripts especially sensitive to reduced ribosome concentrations. It is likely that differential p53 responses to ribosome assembly defects and changes in translation profiles caused by limited ribosome production and/or assembly of specialized ribosomes contribute to ribosomopathy phenotypes to varying degrees (Bohnsack and Bohnsack, 2019). DBA patients are predisposed to develop myelodysplastic syndrome (MDS), acute myeloid leukemia, and solid tumors (Vlachos et al., 2012; Golomb et al., 2014). The most common genetic alterations in DBA are found in the RPS19 gene, but mutations and deletions have also been identified in several other RP genes (Narla et al., 2011; Golomb et al., 2014). Haploinsufficiency of RPS14 and RPSA has been detected in 5q syndrome, acquired somatic deletion of chromosome 5q, and isolated congenital asplenia (Vlachos et al., 2012; Golomb et al., 2014), respectively. Recent studies on animal models of ribosomopathies and patients confirm the role of p53 in mediating specific pathological manifestations of these disorders (Bursac et al., 2014; Golomb et al., 2014). Interestingly, X-DC, SDS, CHH, and DBA are characterized by high cancer incidence, supporting the hypothesis that specific qualitative defects in ribosome biogenesis can also induce oncogenesis (Narla and Ebert, 2010). It has been proposed that changes in the complex ribosome structure may be responsible for alterations in mRNA translation relevant to neoplastic transformation. For example, X-DC is caused by mutations in the gene encoding the ubiquitin ligase dyskerin (DKC1) and is characterized by progressive proliferative tissue failure associated with an 11-fold increased risk of malignancy compared to the general population (Narla and Ebert, 2010; Poortinga et al., 2011; Taoka et al., 2018). The DKC1 product, dyskerin, is a key protein involved in small nucleolar ribonucleoproteins (snoRNPs) involved in rRNA processing (Meier, 2006; Kiss et al., 2010; Böğürcü-Seidel et al., 2023). It is also necessary for site-specific conversion of uridine to pseudouridine in rRNA molecules (Meier, 2006; Kiss et al., 2010; Böğürcü-Seidel et al., 2023). Pseudouridines are located within specific ribosome domains important for tRNA and mRNA binding, and loss of pseudouridine incorporation into rRNA may impair translation of specific mRNAs (Strunk et al., 2012; Ghalei et al., 2017; Sharma et al., 2017). Altered rRNA modifications can trigger tumorigenesis or promote oncogenesis. Aberrant expression of small nucleolar RNAs (snoRNAs) affects the methylation process (Elhamamsy et al., 2025; Lafontaine, 2015; Gay et al., 2022). For instance, increased expression of snoRNA SNORD42A, responsible for 2′-O-methylation of U116 in 18S rRNA, is observed in acute myeloid leukemia (AML) patients (Elhamamsy et al., 2025; Marcel et al., 2013). Breast cancer samples show variability in 2′-O-methylation of rRNA, which correlates differentially with tumor subtype and malignancy grade (Elhamamsy et al., 2025; Marcel et al., 2020). When tumor cells experience stress, such as hypoxia, rRNAs acquire different methylation patterns and generate specialized ribosome subpopulations capable of internal ribosome entry site (IRES)-mediated translation (Elhamamsy et al., 2025; Metge et al., 2020). In normal cells, the expression of the key rRNA methyltransferase fibrillarin is regulated by p53. Mutations or alterations in p53 affect fibrillarin levels and activity, resulting in hypermodified rRNA, with 2′-O-methylation occurring at sites normally unmethylated in p53-positive cells (Sharma et al., 2017; Marcel et al., 2018), potentially causing decreased translation accuracy and enhanced IRES-mediated translation of several oncogenes (Elhamamsy et al., 2025; Macias et al., 2010; Marcel et al., 2013; 2018). Thus, the paradoxical observation that RP misregulation characterized by hypoproliferation leads to hyperproliferative disease such as cancer has several explanations: selective pressure bypassing p53, direct regulation of oncogenes and tumor suppressors by RPs, and preferential mRNA translation including the existence of specialized “oncoribosomes.” Changes in ribosome concentration may also lead to selective mRNA translation and oncogenesis. Besides p53 homeostasis regulation, aberrant RP expression may cause p53 mutations.

Some ribosome biogenesis factors, such as nucleolin (NCL) (Table 1), also play a role in cancer development and metastasis. NCL is not exclusively localized to the nucleolus; it can also be found in the cytoplasm and on the cell outer membrane. Various ligands are capable of binding to cell surface NCL and influencing the physiological functions of the cell (Chen and Xu, 2016). The interaction of endostatin with NCL on the surface of endothelial cells potentiates an anti-angiogenic response and inhibits tumor growth (Weeks et al., 2019).

Most nervous system pathologies are caused by abnormal protein synthesis, and ribosome dysfunction disrupts the homeostasis of neurons and glial cells (Drokhlyansky et al., 2020; Scheurer et al., 2021; Jiao et al., 2023). Accumulating data indicate that disrupted ribosome biogenesis is involved in aging and neurodegeneration (Jiao et al., 2023). Moreover, dysregulation of ribosome biogenesis is neuropathogenic in the context of the developing nervous system (Hetman and Slomnicki, 2019). Depending on the developmental period/cell type, the pathological consequences can range from microcephaly caused by loss of functions such as ID or ASD to neurodegeneration (Hetman and Slomnicki, 2019). In recent years, there has been an increase in the number of diseases identified as novel congenital ribosomopathies associated with severe developmental pathologies. These extremely rare disorders are characterized by mutations in ribosomal proteins or in factors involved in ribosome biogenesis (Venturi and Montanaro, 2020). These ribosomopathies are heterogeneous diseases, manifesting as generalized multisystemic symptoms or more specific manifestations selective for a single tissue or organ. A striking example of a multisystemic ribosomopathy is the rare and severe Bowen-Conradi syndrome (BCS), which is characterized by intellectual disability, microcephaly, micrognathia, a prominent nose, rocker-bottom feet, and flexion contractures of the joints (Lowry et al., 2003; Armistead et al., 2014; 2015; Venturi and Montanaro, 2020), and which leads to death in early childhood. Its cause is a specific mutation in the gene for the Essential for Mitotic Growth 1 (EMG1) protein. EMG1 is a methyltransferase necessary for the proper maturation of 18S rRNA (Armistead et al., 2014). The mutation leads to protein destabilization and a substantial decrease in its levels in patient-derived fibroblasts and lymphoblasts. Furthermore, the mutation causes a delay in the processing of the 18S rRNA precursor and disrupts the normal intracellular localization of the mutant EMG1 protein: its recruitment to the nucleolus is reduced, while it accumulates in abnormal nuclear foci and is subsequently degraded via the proteasome (Warda et al., 2016; Menjivar-Vallecillo et al., 2025). This results in significantly reduced proliferation and a G2/M phase arrest, which impedes normal cell entry into mitosis. Interestingly, however, steady-state levels of mature 18S rRNA and overall protein synthesis rates may remain unchanged (Armistead et al., 2014; 2015).

Tissues with rapid proliferation, such as the neuroepithelium of the developing embryo where EMG1 expression is particularly high, demonstrate a special vulnerability to EMG1 dysfunction. The reduced proliferation of neuroepithelial cells and the underlying mesenchyme likely explains the severe nervous system malformations in patients and the impaired neural tube closure—the precursor to the central nervous system—observed in mouse models (Armistead et al., 2015). This developmental defect may be directly linked to the loss of EMG1’s methyltransferase activity, given the established importance of methylation processes (particularly the folate cycle) for neurulation. As a potential therapeutic approach for BCS, the use of S-adenosylmethionine (SAM)—a universal methyl group donor and a cofactor for EMG1—is being considered (Armistead et al., 2015).

The nucleolus is not just a “factory” for assembling ribosomes, but a dynamic and multifunctional cellular regulatory hub, influencing key processes from cell cycle control and proliferation to decisions regarding differentiation or cell death. Consequently, dysfunction in nucleolar structure and activity is frequently central to severe pathologies, particularly oncological and neurodegenerative disorders, highlighting its critical role in maintaining normal cellular physiology.

At the core of this intricate regulatory network are two key opposing factors: the MYC oncogene and the p53 tumor suppressor. MYC acts as a master driver of cellular growth, potently stimulating the production of all ribosomal components, thereby directing the cell toward division. MYC coordinately enhances the activity of all three RNA polymerases. However, critical questions regarding its function remain open. For instance, how is the balance precisely regulated between MYC’s pro-oncogenic activity and its ability to activate suppressor pathways through excessive ribosome biogenesis stimulation? What are the subtle mechanisms determining whether MYC overexpression leads to uncontrolled proliferation or, conversely, to the activation of p53-dependent apoptosis?

In contrast to MYC, p53 is activated in response to various stresses, including DNA damage or disruptions in the ribosome assembly process itself. p53 activation leads to cell cycle arrest or apoptosis, preventing the division of damaged cells. However, the mechanism of its activation specifically during nucleolar stress remains a subject of debate and is represented by two primary, non-mutually exclusive models.

The first model, relocalization, posits that stress causes nucleolar structural reorganization, resulting in the translocation of key proteins, such as ribosomal proteins RPL5 and RPL11, into the nucleoplasm. There, they bind to MDM2 and directly inhibit its ability to ubiquitinate p53 for degradation, leading to the accumulation of active p53. The second model, direct nucleolar control, focuses on the nucleolus normally serving as a specialized compartment for efficient p53 ubiquitination. According to this model, stress does not necessarily cause massive protein export but rather disrupts the very organization of p53 ubiquitination within the nucleolus allowing p53 exit in a stable form.

It is challenging to definitively prove that the observed relocalization of RPL11/RPL5 is the cause rather than a consequence of p53 stabilization or general nucleolar restructuring. Not all studies observe massive ribosomal protein release before p53 stabilization. The release of ribosomal proteins in the first model could simultaneously trigger nucleolar homeostasis disruptions described by the second model. Both processes may operate in parallel, amplifying the response. The shuttling of p53/MDM2 through the nucleolus and the ubiquitination process are extremely rapid and dynamic, making them technically difficult to capture and measure without perturbing the entire system.

Both models primarily focus on MDM2. However, other E3 ubiquitin ligases for p53 exist, alongside alternative degradation pathways (e.g., calpain-dependent). Their potential contribution under stress conditions, which may vary, is seldom accounted for. Furthermore, the oligomeric state of p53 critically influences its degradation, localization, and activity. Neither model fully explains how nucleolar stress affects this status and how it integrates into the activation mechanism. The hypothesis regarding oligomer transition within the nucleolus remains unconfirmed. Determining which model predominates under different stress conditions or how they cooperatively amplify the signal is a significant unresolved challenge.

The search for and study of p53-independent mechanisms controlling the cell cycle in response to ribosome assembly defects is of particular importance. Several alternative pathways have been identified, such as those involving stabilization of cyclin-dependent kinase inhibitors or suppression of transcription factors. However, it remains incompletely resolved which mechanism predominates under various stress conditions. Understanding these subtle mechanisms could form the basis for developing therapies against tumors where the canonical p53 pathway is already dysfunctional.

A crucial aspect of modern understanding is the recognition of ribosome heterogeneity, stemming from variations in rDNA sequences, tissue-specific post-transcriptional rRNA modifications, differences in ribosomal protein composition, and their post-translational modifications. A key unresolved question is whether cells purposefully produce distinct ribosome populations for optimal translation of specific mRNA sets depending on tissue type, developmental stage, or external conditions—supported by evidence of tissue-specific ribosome variants, clear ribosomopathy phenotypes, and links between specific modifications and the translation of particular transcripts. Alternatively, ribosome heterogeneity may be a byproduct of inaccuracies in the biogenesis process, lacking significant functional relevance, with observed effects (as in ribosomopathies) explained by a general reduction in ribosome quantity/quality, activation of stress pathways, and global translational impairment. The signal for specialization might be statistically weak against the background of the overall ribosome pool.

To date, primarily correlations between specific variant expression and tissue types have been established. It is not proven that these variants are causally necessary and sufficient for shaping a tissue-specific proteome. It is extremely difficult to isolate and study the functional properties of pure ribosome populations containing only one specific rRNA variant in a living cell. Separating the effect of an rRNA variant from confounding factors—such as specific ribosomal protein sets, modifications, and translational factor activities—is challenging. How is the transcription of specific rDNA variants regulated in a given cell? What epigenetic marks or specific factors govern this? Furthermore, it is unclear what contributes more to ribosome “specialization”—rRNA sequence, its modifications, or ribosomal protein composition? How are these regulatory layers coordinated?

Regardless of the precise mechanism of p53 activation and the functional significance of ribosome heterogeneity, the ultimate outcome of system imbalance is malignant transformation. This often arises from MYC hyperactivation or p53 function loss, making the ribosome biogenesis process itself a vulnerable and promising therapeutic target. The investigation of p53-independent cell cycle control mechanisms activated in response to disrupted ribosome assembly is thus of paramount importance.

In conclusion, ongoing research continues to unravel the molecular complexity of the nucleolus. Understanding the dynamic balance between MYC and p53, the mechanisms of the stress response, and the implications of ribosome heterogeneity not only deepens our fundamental knowledge of cell biology but also paves the way for developing novel, more targeted therapeutic strategies against diseases rooted in nucleolar dysfunction, particularly cancer.