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Results for “Ribosomal stress”

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At least 19 recordsLinked to original sources

PubMed2006

MDMX regulation of p53 response to ribosomal stress.

Ribosomal stress such as disruption of rRNA biogenesis activates p53 by release of ribosomal proteins from the nucleoli, which bind to MDM2 and inhibit p53 degradation. We found that p53 activation by ribosomal stress requires degradation of MDMX in an MDM2-dependent fashion. Tumor cells overexpressing MDMX are less sensitive to actinomycin D-induced growth arrest due to formation of inactive p53-MDMX complexes. Knockdown of MDMX increases sensitivity to actinomycin D, whereas MDMX overexpression abrogates p53 activation and prevents growth arrest. Furthermore, MDMX expression promotes resistance to the chemotherapeutic agent 5-fluorouracil (5-FU), which at low concentrations activates p53 by inducing ribosomal stress without significant DNA damage signaling. Knockdown of MDMX abrogates HCT116 tumor xenograft formation in nude mice. MDMX overexpression does not accelerate tumor growth but increases resistance to 5-FU treatment in vivo. Therefore, MDMX is an important regulator of p53 response to ribosomal stress and RNA-targeting chemotherapy agents.

Animals↗

PubMed2026

Comparative toxicoproteomics reveals distinct proteostasis and ribosomal stress signatures of CMIT and PHMG.

Methylchloroisothiazolinone (CMIT) and polyhexamethylene guanidine (PHMG) are antimicrobial biocides associated with pulmonary toxicity, although their comparative cellular stress mechanisms remain unclear. Here, we investigated how CMIT and PHMG differentially alter the proteome of human alveolar epithelial A549 cells under subcytotoxic conditions. Cells were exposed to CMIT or PHMG, and global proteomic profiling was performed using label-free liquid chromatography-tandem mass spectrometry. Differentially expressed proteins (DEPs) were identified at a 1% false discovery rate with an absolute log2 fold change ≥1. Functional analyses were conducted using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Ingenuity Pathway Analysis, and selected proteins were validated by western blotting. Comparative toxicoproteomics revealed distinct stress-response signatures induced by the two biocides. CMIT preferentially altered proteins associated with proteostasis, oxidative stress, and protein quality control, whereas PHMG was characterized by coordinated depletion of ribosome-associated and translation-related proteins. A total of 73 and 155 DEPs were identified in CMIT- and PHMG-treated cells, respectively, with 22 proteins shared between treatments. Western blotting confirmed PSMD3, TUBB2A, and GLRX1 as CMIT-responsive proteins and THRAP3, DHX15, and RPL4 as PHMG-responsive markers. These findings provide comparative mechanistic insight into how CMIT and PHMG induce distinct epithelial stress responses and identify candidate protein markers that may support future in vitro assessment of biocide-induced pulmonary toxicity.

CMIT↗

PubMed2003

Requirement for SAPK-JNK signaling in the induction of apoptosis by ribosomal stress in REH lymphoid leukemia cells.

The present studies examined performance of SAPK cascades and apoptotic commitment following ribosomal trauma in REH lymphoid leukemia cells. Ribostatic insults included disruption of ribosomal activity by mechanistically dissimilar agents such as blasticidin-S (BCS) (which binds 28S-rRNA to block peptidyl bond formation), kasugamycin (KSM) (which binds 18S-rRNA to prevent translational initiation), and cycloheximide (CHX) (which blocks A-site to P-site translocation of peptidyl-tRNA). Exposure of REH cells to BCS elicited DNA degradation and apoptotic cytolysis. BCS stimulated JNK1/JNK2 and p38, and their shared targets c-Jun and ATF2. Inhibition of JNK1/JNK2 (but not of p38) antagonized blasticidin-induced apoptosis, whereas targeting alternative ribosomal sites with KSM or CHX limited translation, but failed to activate the SAPK cascade or initiate apoptosis. Our findings indicate that interference with 28S-rRNA by BCS initiates apoptosis in REH cells through recruitment of SAPK-JNK signaling. Disparities between the lethal actions of BCS, KSM, and CHX appear to reflect established differences in the subribosomal targets of these agents. We propose that the SAPK cascade comprises an essential mechanism for the transduction of specific lethal stress signals emanating from active ribosomes, and that interference with the 28S-rRNA, rather than the peptidyl transfer center of the large subunit, is critical to apoptotic commitment.

Apoptosis↗

PubMed2006

Ribosomal stress couples the unfolded protein response to p53-dependent cell cycle arrest.

Protein misfolding in the endoplasmic reticulum (ER) triggers a signaling pathway termed the unfolded protein response path-way (UPR). UPR signaling is transduced through the transmembrane ER effectors PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE-1), and activating transcription factor 6 (ATF6). PERK activation triggers phosphorylation of eIF2alpha leading to repression of protein synthesis, thereby relieving ER protein load and directly inhibiting cyclin D1 translation thereby contributing to cell cycle arrest. However, PERK(-/-) murine embryonic fibroblasts have an attenuated G(1)/S arrest that is not attributable to cyclin D1 loss, suggesting a cyclin D1-independent mechanism. Here we show that the UPR triggers p53 accumulation and activation. UPR induction promotes enhanced interaction between the ribosome proteins (rpL5, rpL11, and rpL23) and Hdm2 in a PERK-dependent manner. Interaction with ribosomal proteins results in inhibition of Hdm2-mediated ubiquitination and degradation of p53. Our data demonstrate that ribosomal subunit:Hdm2 association couples the unfolded protein response to p53-dependent cell cycle arrest.

Cell Cycle↗

PubMed2003

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.

Amino Acid Sequence↗

PubMed2007

The influence of ribosome modulation factor on the survival of stationary-phase Escherichia coli during acid stress.

Ribosome modulation factor (RMF) was shown to have an influence on the survival of Escherichia coli under acid stress during stationary phase, since the viability of cultures of a mutant strain lacking functional RMF decreased more rapidly than that of the parent strain at pH 3. Loss of ribosomes was observed in both strains when exposed to low pH, although this occurred at a higher rate in the RMF-deficient mutant strain, which also suffered from higher levels of rRNA degradation. It was concluded that the action of RMF in limiting the damage to rRNA contributed to the protection of E. coli under acid stress. Expression of the rmf gene was lower during stationary phase after growth in acidified media compared to media containing no added acid, and the increased rmf expression associated with transition from exponential phase to stationary phase was much reduced in acidified media. It was demonstrated that RMF was not involved in the stationary-phase acid-tolerance response in E. coli by which growth under acidic conditions confers protection against subsequent acid shock. This response was sufficient to overcome the increased vulnerability of the RMF-deficient mutant strain to acid stress at pH values between 6.5 and 5.5.

Acids↗

PubMed2026

Nanopore Sequencing Reveals rRNA Modification Changes in Human Cells Experiencing Oxidative or Inflammatory Stress.

Ribosomal RNA (rRNA) modifications are tuned to regulate protein synthesis; however, their temporal dynamics during oxidative or inflammatory stress remain poorly understood. Nanopore direct RNA sequencing using Dorado v5.2.0 modification-aware models for the data analysis was employed to map human rRNA epitranscriptomic marks in a cell line undergoing oxidative stress, inflammatory stress, or ferroptosis. Oxidative stress triggered a global trend of decreased modification occupancy in which six modifications shifted significantly over 48 h, particularly, 18S Ψ573 and 18S m6A1832. Conversely, inflammatory stress induced a complex response involving an acute pulse of hypermodifications at 28S Um1773 and 28S Ψ1779, for example, and chronic hypomodification at specific target sites (e.g., 18S Gm1328 and 28S Gm4228). In this work, the pseudouridine modifications 28S Ψ4296 and 28S Ψ4353 were identified as "universal stress markers" that decreased under all stressors studied, including ferroptosis. Mapping these changes onto the ribosome structure revealed that they reside in functional regions such as the decoding center and A-site finger, supporting a role in functional ribosome reprogramming during stress. Analysis of mitochondrial rRNA (mt-rRNA) revealed modification shifts within the peptidyl transferase center, suggesting a mechanism to attenuate mitochondrial translation during chronic stress. This work demonstrates that oxidative and inflammatory stress drive distinct, time-resolved remodeling of the human rRNA epitranscriptome and provides a framework for using rRNA modifications as biomarkers of cellular health during oxidative or inflammatory stress exposure.

Humans↗

PubMed1995

Cell stress and ribosome crystallization.

New methods of inducing ribosome crystals in chick embryos are described, and it is shown that a close relationship exists between cell stress and ribosome crystallization. The formation of ribosome crystals in the nucleus and/or in the cytoplasm depends on the type of treatment and on the temperature of incubation, but in all cases the key event that produces crystallizable ribosomes appears to be a damage to the ribosome transport system. This prevents the movement of messenger-free ribosomes towards their destination sites, and accumulates a pool of potentially crystallizable ribosomes in restricted areas of the cell. This mechanism accounts for the fact that ribosome crystallization occurs in chick embryos in the widely different conditions such as those induced by cooling, anoxia, heat-shocks and programmed cell death.

Animals↗

PubMed2004

Genetic and biochemical interactions among Yar1, Ltv1 and Rps3 define novel links between environmental stress and ribosome biogenesis in Saccharomyces cerevisiae.

In the yeast S. cerevisiae, ribosome assembly is linked to environmental conditions by the coordinate transcriptional regulation of genes required for ribosome biogenesis. In this study we show that two nonessential stress-responsive genes, YAR1 and LTV1, function in 40S subunit production. We provide genetic and biochemical evidence that Yar1, a small ankyrin-repeat protein, physically interacts with RpS3, a component of the 40S subunit, and with Ltv1, a protein recently identified as a substoichiometric component of a 43S preribosomal particle. We demonstrate that cells lacking YAR1 or LTV1 are hypersensitive to particular protein synthesis inhibitors and exhibit aberrant polysome profiles, with a reduced absolute number of 40S subunits and an excess of free 60S subunits. Surprisingly, both mutants are also hypersensitive to a variety of environmental stress conditions. Overexpression of RPS3 suppresses both the stress sensitivity and the ribosome biogenesis defect of Deltayar1 mutants, but does not suppress either defect in Deltaltv1 mutants. We propose that YAR1 and LTV1 play distinct, nonessential roles in 40S subunit production. The stress-sensitive phenotypes of strains lacking these genes reveal a hitherto unknown link between ribosome biogenesis factors and environmental stress sensitivity.

Mutation↗

PubMed2006

Knockdown of ribosomal protein S3 protects human cells from genotoxic stress.

Human ribosomal protein S3 (hS3) has a high apparent binding affinity for the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG). The hS3 ribosomal protein has also been found to inhibit the base excision repair (BER) enzyme hOGG1 from liberating 8-oxoG residing in a 5'-end-labeled oligonucleotide. To understand the in vivo involvement of hS3 in BER, we have turned to RNA interference to generate knockdown of hS3 in cells exposed to DNA damaging agents. Here we show that a 40% knockdown of hS3 resulted in as much as a seven-fold increase in the 24h survival-rate of HEK293 cells exposed to hydrogen peroxide. Significant protection to the alkylating agent methyl methanesulfonate (MMS) was also observed. Protection to the chemotherapeutic alkylating agent Thio-TEPA was only revealed at longer exposure times where the agent became more toxic to untransfected human cells. Overall, these results raise the possibility that hS3 interferes with the repair of the DNA lesions produced by genotoxic agents that potentially could play a role in the onset of cancer and other pathological states such as aging.

Alkylating Agents↗

PubMed2000

Creation of a stress-activated p90 ribosomal S6 kinase. The carboxyl-terminal tail of the MAPK-activated protein kinases dictates the signal transduction pathway in which they function.

Mitogen-activated protein kinase-activated protein kinases (MAPKAPKs) lie immediately downstream of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), and p38 MAPK. Although the family of MAPKAPKs shares sequence similarity, it demonstrates selectivity for the upstream activator. Here we demonstrate that each of the ERK- and p38 MAPK-regulated MAPKAPKs contains a MAPK docking site positioned distally to the residue(s) phosphorylated by MAPKs. The isolated MAPK docking sites show specificity for the upstream activator similar to that reported for the full-length proteins. Moreover, replacement of the ERK docking site of p90 ribosomal S6 kinase with the p38 MAPK docking site of MAPKAPK2 converts p90 ribosomal S6 kinase into a stress-activated kinase in vivo. It is apparent that mechanisms controlling events downstream of the proline-directed MAPKs involve specific MAPK docking sites within the carboxyl termini of the MAPKAPKs that determine the cascade in which the MAPKAPK functions.

Amino Acid Sequence↗

PubMed2025

Mapping transcriptional responses to cellular perturbation dictionaries with RNA fingerprinting.

Single-cell perturbation dictionaries provide systematic measurements of how cells respond to genetic and chemical perturbations, and create the opportunity to assign causal interpretations to observational data. Here, we introduce RNA fingerprinting, a statistical framework that maps transcriptional responses from new experiments onto reference perturbation dictionaries. RNA fingerprinting learns denoised perturbation "fingerprints" from single-cell data, then probabilistically assigns query cells to one or more candidate perturbations while accounting for uncertainty. We benchmark our method across ground-truth datasets, demonstrating accurate assignments at single-cell resolution, scalability to genome-wide screens, and the ability to resolve combinatorial perturbations. We demonstrate its broad utility across diverse biological settings: identifying context-specific regulators of p53 under ribosomal stress, characterizing drug mechanisms of action and dose-dependent off-target effects, and uncovering cytokine-driven B cell heterogeneity during secondary influenza infection in vivo. Together, these results establish RNA fingerprinting as a versatile framework for interpreting single-cell datasets by linking cellular states to the underlying perturbations which generated them.

Journal Article↗

PubMed2005

Rck2 is required for reprogramming of ribosomes during oxidative stress.

Rck2 is a mitogen-activated protein kinase-activated protein kinase in yeast implicated in translational regulation. rck2Delta mutants are mildly sensitive to oxidative stress, a condition that causes dissociation of actively translating ribosomes (polysomes). In rck2Delta cells, polysomes are lost to an even higher degree than in the wild-type upon stress. Cells overexpressing the catalytically inactive rck2-kd allele are highly sensitive to oxidative stress. In such cells, dissociation of polysomes upon stress was instead greatly delayed. The protein synthesis rate decreased to a similar degree as in wild-type cells, however, indicating that in rck2-kd cells, the polysome complexes were inactive. Array analyses of total and polysome-associated mRNAs revealed major deregulation of the translational machinery in rck2 mutant cells. This involves transcripts for cytosolic ribosomal proteins and for processing and assembly of ribosomes. In rck2Delta cells, weakly transcribed mRNAs associate more avidly with polysomes than in wild-type cells, whereas the opposite holds true for rck2-kd cells. This is consistent with perturbed regulation of translation elongation, which is predicted to alter the ratio between mRNAs with and without strong entry sites at ribosomes. We infer that imbalances in the translational apparatus are a major reason for the inability of these cells to respond to stress.

Alleles↗

PubMed1990

Hypoxic stress-induced changes in ribosomes of maize seedling roots.

The hypoxic stress response of Zea mays L. seedling roots involves regulation of gene expression at transcriptional and posttranscriptional levels. We investigated the effect of hypoxia on the translational machinery of seedling roots. The levels of monoribosomes and ribosomal subunits increased dramatically within 1 hour of stress. Prolonged hypoxia resulted in continued accumulation of nontranslating ribosomes, as well as increased levels of small polyribosomes. The return of seedlings to normal aerobic conditions resulted in recovery of normal polyribosome levels. Comparison of ribosomal proteins from control and hypoxic roots revealed differences in quantity and electrophoretic mobility. In vivo labeling of roots with [(35)S]methionine revealed variations in newly synthesized ribosomal proteins. In vivo labeling of roots with [(32)P]orthophosphate revealed a major reduction in the phosphorylation of a 31 kilodalton ribosomal protein in hypoxic stressed roots. In vitro phosphorylation of ribosomal proteins by endogenous kinases was used to probe for differences in ribosome structure and composition. The patterns of in vitro kinased phosphoproteins of ribosomes from control and hypoxic roots were not identical. Variation in phosphoproteins of polyribosomes from control and hypoxic roots, as well as among polyribosomes from hypoxic roots were observed. These results indicate that modification of the translational machinery occurs in response to hypoxic stress.

Journal Article↗

PubMed2026

Programmed ribosomal frameshifting triggers translational stress to promote viral replication.

Programmed ribosomal frameshifting (PRF) is a conserved viral strategy for expressing polyproteins from compact genomes. Although PRF is traditionally viewed as a structural mechanism, here we show that it functions as a regulatory signal that rewires host translation in favor of viral replication. A minimal SARS-CoV-2 PRF element is sufficient to activate the GCN2 arm of the integrated stress response (ISR) independently of the canonical ISR sensor ZAKα. This activation serves as a temporal switch during early infection to shut off host translation and is required for viral propagation in cells and human airway organoids. Proteomic and genetic screens identify DRG1 and IGF2BP3 as key mediators of PRF-induced GCN2 activation. We further show that this PRF-GCN2 axis is conserved in human immunodeficiency virus (HIV)-1 and West Nile virus, highlighting its broad relevance across RNA viruses. These findings reveal a sophisticated mechanism of viral translational control, highlighting PRF as a stress-inducing module that enhances viral replication.

RNA virus↗

PubMed2006

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation.

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2alpha phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2alpha phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2alpha to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2alpha phosphorylation-dependent and -independent pathways that target translation initiation.

Antigens, Surface↗

PubMed2005

The thioredoxin system protects ribosomes against stress-induced aggregation.

We previously showed that thioredoxins are required for dithiothreitol (DTT) tolerance, suggesting they maintain redox homeostasis in response to both oxidative and reductive stress conditions. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to DTT to identify cell functions involved in resistance to reductive stress. We identified 195 mutants, whose gene products are localized throughout the cell. DTT-sensitive mutants were distributed among most major biological processes, but they particularly affected gene expression, metabolism, and the secretory pathway. Strikingly, a mutant lacking TSA1, encoding a peroxiredoxin, showed a similar sensitivity to DTT as a thioredoxin mutant. Epistasis analysis indicated that thioredoxins function upstream of Tsa1 in providing tolerance to DTT. Our data show that the chaperone function of Tsa1, rather than its peroxidase function, is required for this activity. Cells lacking TSA1 were found to accumulate aggregated proteins, and this was exacerbated by exposure to DTT. Analysis of the protein aggregates revealed that they are predominantly composed of ribosomal proteins. Furthermore, aggregation was found to correlate with an inhibition of translation initiation. We propose that Tsa1 normally functions to chaperone misassembled ribosomal proteins, preventing the toxicity that arises from their aggregation.

DNA-Binding Proteins↗

PubMed2001

HSP70-mediated acceleration of translational recovery after stress is independent of ribosomal RNA synthesis.

HSP70 is known to protect cells against stressful events. In the present study, the hypothesis was investigated that elevated HSP70 levels protect RNA polymerase I during stress, leading to decreased inhibition of ribosomal RNA (rRNA) synthesis and accelerated recovery of protein translation after stress. To this end, transcriptional and translational activity was studied in H9c2 cells during recovery after a severe heat treatment (SHT, 1 h 45 degrees C) in the presence of elevated HSP70 levels. The latter was achieved by heat pretreatment or by adenovirus-mediated hsp70 gene transfer. Rates of transcription and translation were determined by measuring cellular 3H-labelled uridine and leucine incorporation, respectively. The two types of pretreatment did not affect basal rates of transcription and translation, immediately before SHT. During SHT, both transcriptional and translational rates dropped to less than 10% of basal levels in pretreated as well as non-pretreated cells. Two and four h after SHT, both transcriptional and translational rates were significantly higher in HSP70-overexpressing cells compared to non-pretreated cells. However, immediately after SHT, transcription rates were similarly depressed in non-pretreated and pretreated cells, showing that increased levels of HSP70 did not protect RNA polymerase I activity during SHT. Thus, the HSP70-mediated acceleration of translational recovery is not preceded in time by an enhanced recovery of rRNA synthesis. Therefore, the HSP70-mediated early recovery of protein synthesis after heat stress is independent of rRNA synthesis.

Adenoviridae↗