Ribosomal RNA Processing Research Articles

Heterologous Expression, Purification and Structural Characterization of Ribonuclease E from Mycobacterium smegmatis

Rapid adaptations to various environmental changes are key to the survival of bacterial cells, which is mediated at the level of transcription initiation and RNA degradation/processing. Ribonuclease E (RNase E), an essential endoribonuclease gene in Mycobacterium species, is a key mediator in most reactions of RNA metabolism such as messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA) processing and their degradation qualifying it as an important drug target. Here, we first report the cloning of full-length Mycobacterium smegmatis mc2155 (M. smegmatis) RNase E [MSMEG_4626] and its expression with isopropyl β-D-1-thiogalactopyranoside (IPTG) in Escherichia coli (E. coli) as a glutathione S-transferase (GST) fusion protein. The best expression of the recombinant protein was optimized with respect to different parameters, such as E. coli strains, temperature, IPTG concentrations and time points of induction. Despite the use of protease inhibitors, we observed low-molecular-weight degradation products along with full-length fusion proteins. Maximum expression of soluble and least degraded full-length RNase E was obtained at 32 °C/0.1-mM IPTG, further purified by affinity chromatography using glutathione agarose beads and confirmed by immunoblotting technique using anti-GST antibody. In silico characterization using multiple sequence alignment, homology modeling and docking studies revealed four amino acid residues, Asp613, Phe657, Ile662 and Glu688, in the catalytic domain of RNase E that are completely conserved in both E. coli and Mycobacterium species, also involved in RNA binding and might act as candidate drug targets to combat antibiotic-resistant mycobacterial diseases. Additionally, recombinant GST-RNase E protein can unravel the mechanism of RNA degradation/processing through protein–protein interaction studies.

From the archives: regulation of mRNA translation, epigenetic machinery components, and processing of a composite large subunit ribosomal RNA.

From the archives: regulation of mRNA translation, epigenetic machinery components, and processing of a composite large subunit ribosomal RNA.

Therapeutic targeting of CNBP phase separation inhibits ribosome biogenesis and neuroblastoma progression via modulating SWI/SNF complex activity

Neuroblastoma (NB) is the most common extracranial malignancy in childhood; however, the mechanisms underlying its aggressive characteristics still remain elusive. Integrative data analysis was performed to reveal tumour-driving transcriptional regulators. Co-immunoprecipitation and mass spectrometry assays were applied for protein interaction studies. Real-time reverse transcription-polymerase chain reaction, western blotting, sequential chromatin immunoprecipitation and dual-luciferase reporter assays were carried out to explore gene expression regulation. The biological characteristics of NB cell lines were examined via gain- and loss-of-function assays. For survival analysis, the Cox regression model and log-rank tests were used. Cellular nucleic acid-binding protein (CNBP) was found to be an independent factor affecting NB outcome, which exerted oncogenic roles in ribosome biogenesis, tumourigenesis and aggressiveness. Mechanistically, karyopherin subunit beta 1 (KPNB1) was responsible for nuclear transport of CNBP, whereas liquid condensates of CNBP repressed the activity of switch/sucrose-nonfermentable (SWI/SNF) core subunits (SMARCC2/SMARCC1/SMARCA4) via interaction with SMARCC2, leading to alternatively increased activity of SMARCC1/SMARCA4 binary complex in facilitating gene expression essential for 18S ribosomal RNA (rRNA) processing in tumour cells, extracellular vesicle-mediated delivery of 18S rRNA and subsequent M2 macrophage polarisation. A cell-penetrating peptide blocking phase separation and interaction of CNBP with SMARCC2 inhibited ribosome biogenesis and NB progression. High KPNB1, CNBP, SMARCC1 or SMARCA4 expression or low SMARCC2 levels were associated with poor survival of NB patients. These findings suggest that CNBP phase separation is a target for inhibiting ribosome biogenesis and tumour progression in NB via modulating SWI/SNF complex activity.

Current research on viral proteins that interact with fibrillarin.

The nucleolus is a multifunctional nuclear domain primarily dedicated to ribosome biogenesis. Certain viruses developed strategies to manipulate host nucleolar proteins to facilitate their replication by modulating ribosomal RNA (rRNA) processing. This association interferes with nucleolar functions resulting in overactivation or arrest of ribosome biogenesis, induction or inhibition of apoptosis, and affecting stress response. The nucleolar protein fibrillarin (FBL) is an important target of some plant and animal viruses. FBL is an essential and highly conserved S-adenosyl methionine (SAM) dependent methyltransferase, capable of rRNA degradation by its intrinsically disordered region (IDR), the glycine/arginine-rich (GAR) domain. It forms a ribonucleoprotein complex that directs 2'-O-methylations in more than 100 sites of pre-rRNAs. It is involved in multiple cellular processes, including initiation of transcription, oncogenesis, and apoptosis, among others. The interaction with animal viruses, including human viruses, triggered its redistribution to the nucleoplasm and cytoplasm, interfering with its role in pre-rRNA processing. Viral-encoded proteins with IDRs as nucleocapsids, matrix, Tat protein, and even a viral snoRNA, can associate with FBL, forcing the nucleolar protein to undergo atypical functions. Here we review the molecular mechanisms employed by animal and human viruses to usurp FBL functions and the effect on cellular processes, particularly in ribosome biogenesis.

Nucleolar URB1 ensures 3' ETS rRNA removal to prevent exosome surveillance.

The nucleolus is the most prominent membraneless condensate in the nucleus. It comprises hundreds of proteins with distinct roles in the rapid transcription of ribosomal RNA (rRNA) and efficient processing within units comprising a fibrillar centre and a dense fibrillar component and ribosome assembly in a granular component1. The precise localization of most nucleolar proteins and whether their specific localization contributes to the radial flux of pre-rRNA processing have remained unknown owing to insufficient resolution in imaging studies2-5. Therefore, how these nucleolar proteins are functionally coordinated with stepwise pre-rRNA processing requires further investigation. Here we screened 200 candidate nucleolar proteins using high-resolution live-cell microscopy and identified 12 proteins that are enriched towards the periphery of the dense fibrillar component (PDFC). Among these proteins, unhealthy ribosome biogenesis 1 (URB1) is a static, nucleolar protein that ensures 3' end pre-rRNA anchoring and folding for U8 small nucleolar RNA recognition and the subsequent removal of the 3' external transcribed spacer (ETS) at the dense fibrillar component-PDFC boundary. URB1 depletion leads to a disrupted PDFC, uncontrolled pre-rRNA movement, altered pre-rRNA conformation and retention of the 3' ETS. These aberrant 3' ETS-attached pre-rRNA intermediates activate exosome-dependent nucleolar surveillance, resulting in decreased 28S rRNA production, head malformations in zebrafish and delayed embryonic development in mice. This study provides insight into functional sub-nucleolar organization and identifies a physiologically essential step in rRNA maturation that requires the static protein URB1 in the phase-separated nucleolus.

Cryo-EM captures early ribosome assembly in action

Ribosome biogenesis is a fundamental multi-step cellular process in all domains of life that involves the production, processing, folding, and modification of ribosomal RNAs (rRNAs) and ribosomal proteins. To obtain insights into the still unexplored early assembly phase of the bacterial 50S subunit, we exploited a minimal in vitro reconstitution system using purified ribosomal components and scalable reaction conditions. Time-limited assembly assays combined with cryo-EM analysis visualizes the structurally complex assembly pathway starting with a particle consisting of ordered density for only ~500 nucleotides of 23S rRNA domain I and three ribosomal proteins. In addition, our structural analysis reveals that early 50S assembly occurs in a domain-wise fashion, while late 50S assembly proceeds incrementally. Furthermore, we find that both ribosomal proteins and folded rRNA helices, occupying surface exposed regions on pre-50S particles, induce, or stabilize rRNA folds within adjacent regions, thereby creating cooperativity.

Inhibition of Ribosomal RNA Processing 15 Homolog (RRP15) Suppressed Tumor Growth, Invasion and Epithelial to Mesenchymal Transition (EMT) of Colon Cancer.

Although ribosomal RNA processing 15 Homolog (RRP15) has been implicated in the occurrence of various cancers and is considered a potential target for cancer treatment, its significance in colon cancer (CC) is unclear. Thus, this present study aims to determine RRP15 expression and biological function in CC. The results demonstrated a strong expression of RRP15 in CC compared to normal colon specimens, which was correlated with poorer overall survival (OS) and disease-free survival (DFS) of the patients. Among the nine investigated CC cell lines, RRP15 demonstrated the highest and lowest expression in HCT15 and HCT116 cells, respectively. In vitro assays demonstrated that the knockdown of RRP15 inhibited the growth, colony-forming ability and invasive ability of the CC cells whereas its overexpression enhanced the above oncogenic function. Moreover, subcutaneous tumors in nude mice showed that RRP15 knockdown inhibited the CC growth while its overexpression enhanced their growth. Additionally, the knockdown of RRP15 inhibited the epithelial-mesenchymal transition (EMT), whereas overexpression of RRP15 promoted the EMT process in CC. Collectively, inhibition of RRP15 suppressed tumor growth, invasion and EMT of CC, and might be considered a promising therapeutic target for treating CC.

Linking molecular-level RNA-protein interactions with mesoscale properties of the nucleolus.

The nucleolus is the most prominent nuclear body, and represents a condensate comprising the machinery of ribosome biogenesis, including multi-step transcription, processing, and assembly of ribosomal RNA (rRNA). These intricate steps are organized in well-characterized nucleolar substructures, with a host of RNA and proteins components that have been described in detail. However, the role of specific molecular-level interactions, such as RNA-RNA and RNA-protein, in governing nucleolar morphology and its material properties is unknown. Towards addressing this problem, we have deployed a suite of chemical biology, quantitative cellular microscopy and in vitro biophysical methods. Specifically, we use chemical perturbations to selectively and transiently inhibit distinct steps of ribosome biogenesis in cells. We then visualize and classify changes in nucleolar morphology as well as profile thermodynamics of nucleolar RNA-binding proteins (RBPs) upon treating cells with these inhibitors. Preliminary results indicate that the RBP thermodynamic profiles and morphologies are dependent on the presence or absence of specific RNA and/or RBP species in the nucleolus, suggesting that select RBP-RNA interactions may differentially contribute to nucleolar structure and function. To test this hypothesis, we are investigating material properties, dynamics, and RNA structural landscapes of in vitro nucleolar condensates with varying RNA and RBP compositions. Given that aberrant ribosome production is a hallmark of cancer and other diseases, our findings on the molecular thermodynamics of the nucleolus will pave the way towards rational therapeutic targeting.

Construction and Validation of A Novel Predictive Model Based on Chromatin Regulators in Endometrial Cancer

Chromatin regulators (CRs) are substantially associated with the prognosis and immunotherapy responses in multiple cancers. However, comprehensive studies related to the prognostic prediction of CRs in endometrial cancer (UCEC) still remains lacking. Therefore, first, we constructed a prognostic model for UCEC associated with 23 CRs using a univariate Cox regression model as well as the glmnet R package. Then, based on the median risk scores, we separated the UCEC sufferers into the high risk set and low risk set, in which the proportion of sufferers with pathological grade G3 and clinical-stage III–IV is higher in the high risk one. Furthermore, we investigated the association of independent prognostic factors, immunotherapy-related scores, GO, KEGG pathways, and drug sensitivity between both groupings. We identified the high risk set tended to have lower IPS, microsatellite instability, and tumor mutational load. GO and KEGG suggested an enrichment of genes related to ribosome biogenesis, RNA processing and metabolism, viral defence, translational regulation, and calmodulin-binding in the high-risk one, while the other one related to cilia movement, microtubule movement, axonal components, and calmodulin-binding. Drug sensitivity assay showed sensitive doxorubicin could be applied to the high-risk patients. All in all, our team successfully built and validated a predictive model for CRs that predicts the overall survival of UCEC patients, which may provide a potential new direction for prognosis prediction and therapeutic target selection for UCEC patients.

Cinchonine-induced cell death in pancreatic cancer cells by downregulating RRP15.

Pancreatic cancer is characterized by poor prognosis and high mortality, while its treatment remains unsatisfactory. Cinchonine, a natural compound present in cinchona bark, is a potential anticancer drug. Whether cinchonine is of relevance to pancreatic cancer therapeutics is unclear. This research showed that the ribosomal RNA-processing 15 homolog (RRP15) expression is decreased in the pancreatic cancer, and RRP15 knockdown inhibited autophagy, and caused apoptosis in pancreatic cancer cells. Cinchonine treatment inhibits the expression of RRP15 and autophagy, and caused apoptosis by leading to the activation of Nrf2 axis in pancreatic cancer cells. Taken together, the above results indicate that cinchonine treatment inhibited autophagy and induced apoptosis through activating Nrf2 axis by downregulating RRP15 in pancreatic cancer cells.

Nuclear α-Synuclein-Derived Cytotoxic Effect via Altered Ribosomal RNA Processing in Primary Mouse Embryonic Fibroblasts.

α-Synuclein (αSyn) is an important player in Parkinson's disease (PD) pathogenesis. The aggregation of αSyn is mainly formed in the cytoplasm, whereas some αSyn accumulation has also been found in the nuclei of neurons. To assess the effect of nuclear αSyn, we generated αSyn conjugated with a nuclear export signal (NES) or a nuclear localization signal (NLS), and compared them with wild-type αSyn in primary mouse embryonic fibroblasts (MEF) using DNA transfection. Overexpression of NLS-αSyn increased cytotoxicity. The levels of apoptotic markers were increased by NLS-αSyn in MEF. Interestingly, an increase in the levels of 40S ribosomal protein 15 was observed in MEF expressing NLS-αSyn. These MEF also showed a higher 28S/18S rRNA ratio. Intriguingly, the expression of NLS-αSyn in MEF enhanced segmentation of nucleolin (NCL)-positive nucleolar structures. We also observed that the downregulation of NCL, using shRNA, promoted a relatively higher 28S/18S rRNA ratio. The reduction in NCL expression accelerated the accumulation of αSyn, and NCL transfection enhanced the degradation of αSyn. These results suggest that nuclear αSyn contributes to the alteration in ribosomal RNA processing via NCL malfunction-mediated nucleolar segmentation, and that NCL is a key factor for the degradation of αSyn.

Mitoribosome Biogenesis.

Mitoribosome biogenesis is a complex and energetically costly process that involves RNA elements encoded in the mitochondrial genome and mitoribosomal proteins most frequently encoded in the nuclear genome. The process is catalyzed by extra-ribosomal proteins, nucleus-encoded assembly factors that act in all stages of the assembly process to coordinate the processing and maturation of ribosomal RNAs with the hierarchical association of ribosomal proteins. Biochemical studies and recent cryo-EM structures of mammalian mitoribosomes have provided hints regarding their assembly. In this general concept chapter, we will briefly describe the current knowledge, mainly regarding the mammalian mitoribosome biogenesis pathway and factors involved, and will emphasize the biological sources and approaches that have been applied to advance the field.

Nucleolar Stress Response via Ribosomal Protein L11 Regulates Topoisomerase Inhibitor Sensitivity of P53-Intact Cancers.

Nucleolar stress response is caused by perturbations in ribosome biogenesis, induced by the inhibition of ribosomal RNA processing and synthesis, as well as ribosome assembly. This response induces p53 stabilization and activation via ribosomal protein L11 (RPL11), suppressing tumor progression. However, anticancer agents that kill cells via this mechanism, and their relationship with the therapeutic efficiency of these agents, remain largely unknown. Here, we sought to investigate whether topoisomerase inhibitors can induce nucleolar stress response as they reportedly block ribosomal RNA transcription. Using rhabdomyosarcoma and rhabdoid tumor cell lines that are sensitive to the nucleolar stress response, we evaluated whether nucleolar stress response is associated with sensitivity to topoisomerase inhibitors ellipticine, doxorubicin, etoposide, topotecan, and anthracyclines. Cell proliferation assay indicated that small interfering RNA-mediated RPL11 depletion resulted in decreased sensitivity to topoisomerase inhibitors. Furthermore, the expression of p53 and its downstream target proteins via western blotting showed the suppression of p53 pathway activation upon RPL11 knockdown. These results suggest that the sensitivity of cancer cells to topoisomerase inhibitors is regulated by RPL11-mediated nucleolar stress responses. Thus, RPL11 expression may contribute to the prediction of the therapeutic efficacy of topoisomerase inhibitors and increase their therapeutic effect of topoisomerase inhibitors.

Structural basis of human SNAPc recognizing proximal sequence element of snRNA promoter

In eukaryotes, small nuclear RNAs (snRNAs) function in many fundamental cellular events such as precursor messenger RNA splicing, gene expression regulation, and ribosomal RNA processing. The snRNA activating protein complex (SNAPc) exclusively recognizes the proximal sequence element (PSE) at snRNA promoters and recruits RNA polymerase II or III to initiate transcription. In view that homozygous gene-knockout of SNAPc core subunits causes mouse embryonic lethality, functions of SNAPc are almost housekeeping. But so far, the structural insight into how SNAPc assembles and regulates snRNA transcription initiation remains unclear. Here we present the cryo-electron microscopy structure of the essential part of human SNAPc in complex with human U6-1 PSE at an overall resolution of 3.49 Å. This structure reveals the three-dimensional features of three conserved subunits (N-terminal domain of SNAP190, SNAP50, and SNAP43) and explains how they are assembled into a stable mini-SNAPc in PSE-binding state with a “wrap-around” mode. We identify three important motifs of SNAP50 that are involved in both major groove and minor groove recognition of PSE, in coordination with the Myb domain of SNAP190. Our findings further elaborate human PSE sequence conservation and compatibility for SNAPc recognition, providing a clear framework of snRNA transcription initiation, especially the U6 system.

Therapeutic metformin concentrations positively regulate proliferation in endometrial epithelial cells (EECs) via mTOR activation and augmented mitochondrial strength.

Metformin, an antidiabetic drug, has recently been repositioned in the treatment of several nondiabetic disorders including reproductive disorders such as polycystic ovarian syndrome (PCOS), where it improves endometrial functions. In vitro studies employing supratherapeutic concentrations (5-20 mM) of metformin, have reported anti-proliferative effects on endometrial epithelial and stromal cells. However, animal and human studies have revealed that therapeutic serum concentrations of metformin range between 20-70 µM. In the present study, the effect of therapeutic concentrations of metformin was studied on endometrial epithelial cells (EECs). Therapeutic concentrations of metformin induced proliferation in Ishikawa and HEC-1A cells. The proliferation of EECs was found mTOR dependent. Interestingly, therapeutic metformin concentrations were not able to activate the classical AMPK signaling. On the contrary, supratherapeutic metformin concentration (10 mM) inhibited mTOR and activated AMPK signaling. Microarray analysis of metformin-treated HEC-1A cells revealed dose-dependent differential effects on biological pathways associated with translation, ribosomal RNA processing, mitochondrial translation and cell proliferation. Therapeutic concentrations of metformin upregulated mitochondrial number as demonstrated by increased MitotrackerTM Red staining and enhanced succinate dehydrogenase (SDHD) expression; however higher concentration (10 mM) abrogated the same. Our results suggest that therapeutic concentrations of metformin augment mitochondrial strength and induce mTOR dependent endometrial cell proliferation.

The ribosomal RNA processing 1B:protein phosphatase 1 holoenzyme reveals non-canonical PP1 interaction motifs.

The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of substrates by associating with >200 regulatory proteins to form specific holoenzymes. The major PP1 targeting protein in the nucleolus is RRP1B (ribosomal RNA processing 1B). In addition to selectively recruiting PP1β/PP1γ to the nucleolus, RRP1B also has a key role in ribosome biogenesis, among other functions. How RRP1B binds PP1 and regulates nucleolar phosphorylation signaling is not yet known. Here, we show that RRP1B recruits PP1 via established (RVxF/SILK/ΦΦ) and non-canonical motifs. These atypical interaction sites, the PP1β/γ specificity, and N-terminal AF-binding pockets rely on hydrophobic interactions that contribute to binding and, via phosphorylation, regulate complex formation. This work advances our understanding of PP1 isoform selectivity, reveals key roles of N-terminal PP1 residues in regulator binding, and suggests that additional PP1 interaction sites have yet to be identified, all of which are necessary for a systems biology understanding of PP1 function.

Exploratory meta-analysis of hypoxic transcriptomes using a precise transcript reference sequence set.

Gene expression studies are intrinsically biased, with many studies influenced by concomitant information such as gene-disease associations. This limitation can be overcome using a data-driven analysis approach without relying on ancillary information. The FANTOM CAGE-Associated Transcriptome project provides a comprehensive meta-assembly of the human transcriptome using coding and noncoding genes. Hypoxia strongly influences gene expression; in addition, noncoding RNA (ncRNA) metabolism is down-regulated in response to hypoxic stimuli. We evaluated the differential response of various transcripts to hypoxia by determining their hypoxia responsiveness scores. Enrichment analysis revealed that several genes associated with ncRNA metabolism, particularly those involved in ribosomal RNA processing, were down-regulated in response to hypoxia. Previously published information from the FANTOM CAGE-Associated Transcriptome project was suitable for meta-analysis of the transcriptome sequencing data from both coding and ncRNAs and to evaluate the hypoxia responsiveness of target transcripts and relationship between sense-antisense transcripts from the same locus. Our results may facilitate functional annotation of various transcripts including ncRNAs, allowing for both sense and antisense and coding and noncoding evaluations.

Multi-Omics Analysis of GNL3L Expression, Prognosis, and Immune Value in Pan-Cancer.

Simple SummaryGuanine nucleotide-binding protein-like 3-like (GNL3L) is a novel GTP-binding nucleolar protein. In this study, we analyzed the expression, prognosis, and immune roles of GNL3L in pan-cancer from multiple omics analyses. The final results showed that GNL3L is differentially expressed in a variety of cancers, plays a prognostic role, and has good immune value. Moreover, GNL3L may affect the occurrence of cancer through processes such as ribonucleoprotein, ribosomal RNA processing, and cell proliferation. At the same time, we established an esophageal cancer (ESCA) prediction model with strong predictive ability and proved that GNL3L can significantly affect the proliferation ability of esophageal cancer cells through clone formation assays. In conclusion, GNL3L is an important biomarker.Guanine nucleotide-binding protein-like 3-like protein (GNL3L) is a novel, evolutionarily conserved, GTP-binding nucleolar protein. This study aimed to investigate the expression, prognosis, and immune value of GNL3L in pan-cancer from multiple omics analyses. Firstly, the expression and prognostic value of GNL3L in pan-cancer were discussed using the TIMER2 database, the GEPIA database, the cBioportal database, COX regression analysis, and enrichment analysis. The association of GNL3L with tumor mutational burden (TMB), tumor microsatellite instability (MSI), mismatch repair (MMR) genes, and immune cells was then analyzed. Finally, an esophageal cancer (ESCA) prediction model was established, and GNL3L clone formation assays were performed. The final results showed that GNL3L is differentially expressed in the vast majority of cancers, is associated with the prognosis of various cancers, and may affect cancer occurrence through processes such as ribonucleoprotein, ribosomal RNA processing, and cell proliferation. At the same time, it was found that the correlation between GNL3L and TMB, MSI, MMR, and various immune cells is significant. The established ESCA prediction model had a strong predictive ability, and GNL3L could significantly affect the proliferation of esophageal cancer cells. In conclusion, GNL3L may serve as an important prognostic biomarker and play an immunomodulatory role in tumors.

Rbp95 binds to 25S rRNA helix H95 and cooperates with the Npa1 complex during early pre-60S particle maturation.

Eukaryotic ribosome synthesis involves more than 200 assembly factors, which promote ribosomal RNA (rRNA) processing, modification and folding, and assembly of ribosomal proteins. The formation and maturation of the earliest pre-60S particles requires structural remodeling by the Npa1 complex, but is otherwise still poorly understood. Here, we introduce Rbp95 (Ycr016w), a constituent of early pre-60S particles, as a novel ribosome assembly factor. We show that Rbp95 is both genetically and physically linked to most Npa1 complex members and to ribosomal protein Rpl3. We demonstrate that Rbp95 is an RNA-binding protein containing two independent RNA-interacting domains. In vivo, Rbp95 associates with helix H95 in the 3′ region of the 25S rRNA, in close proximity to the binding sites of Npa1 and Rpl3. Additionally, Rbp95 interacts with several snoRNAs. The absence of Rbp95 results in alterations in the protein composition of early pre-60S particles. Moreover, combined mutation of Rbp95 and Npa1 complex members leads to a delay in the maturation of early pre-60S particles. We propose that Rbp95 acts together with the Npa1 complex during early pre-60S maturation, potentially by promoting pre-rRNA folding events within pre-60S particles.

Glutamine deficiency in solid tumor cells confers resistance to ribosomal RNA synthesis inhibitors

Ribosome biogenesis is an energetically expensive program that is dictated by nutrient availability. Here we report that nutrient deprivation severely impairs precursor ribosomal RNA (pre-rRNA) processing and leads to the accumulation of unprocessed rRNAs. Upon nutrient restoration, pre-rRNAs stored under starvation are processed into mature rRNAs that are utilized for ribosome biogenesis. Failure to accumulate pre-rRNAs under nutrient stress leads to perturbed ribosome assembly upon nutrient restoration and subsequent apoptosis via uL5/uL18-mediated activation of p53. Restoration of glutamine alone activates p53 by triggering uL5/uL18 translation. Induction of uL5/uL18 protein synthesis by glutamine is dependent on the translation factor eukaryotic elongation factor 2 (eEF2), which is in turn dependent on Raf/MEK/ERK signaling. Depriving cells of glutamine prevents the activation of p53 by rRNA synthesis inhibitors. Our data reveals a mechanism that tumor cells can exploit to suppress p53-mediated apoptosis during fluctuations in environmental nutrient availability.