Diurnal Oscillations in Liver Mass and Cell Size Accompany Ribosome Assembly Cycles.

The liver plays a pivotal role in metabolism and xenobiotic detoxification, processes that must be particularly efficient when animals are active and feed. A major question is how the liver adapts to these diurnal changes in physiology. Here, we show that, in mice, liver mass, hepatocyte size, and protein levels follow a daily rhythm, whose amplitude depends on both feeding-fasting and light-dark cycles. Correlative evidence suggests that the daily oscillation in global protein accumulation depends on a similar fluctuation in ribosome number. Whereas rRNA genes are transcribed at similar rates throughout the day, some newly synthesized rRNAs are polyadenylated and degraded in the nucleus in a robustly diurnal fashion with a phase opposite to that of ribosomal protein synthesis. Based on studies with cultured fibroblasts, we propose that rRNAs not packaged into complete ribosomal subunits are polyadenylated by the poly(A) polymerase PAPD5 and degraded by the nuclear exosome.

RNA Pol II preferentially regulates ribosomal protein expression by trapping disassociated subunits.

RNA polymerase II (RNA Pol II) has been recognized as a passively regulated multi-subunit holoenzyme. However, the extent to which RNA Pol II subunits might be important beyond the RNA Pol II complex remains unclear. Here, fractions containing disassociated RPB3 (dRPB3) were identified by size exclusion chromatography in various cells. Through a unique strategy, i.e., "specific degradation of disassociated subunits (SDDS)," we demonstrated that dRPB3 functions as a regulatory component of RNA Pol II to enable the preferential control of 3' end processing of ribosomal protein genes directly through its N-terminal domain. Machine learning analysis of large-scale genomic features revealed that the little elongation complex (LEC) helps to specialize the functions of dRPB3. Mechanistically, dRPB3 facilitates CBC-PCF11 axis activity to increase the efficiency of 3' end processing. Furthermore, RPB3 is dynamically regulated during development and diseases. These findings suggest that RNA Pol II gains specific regulatory functions by trapping disassociated subunits in mammalian cells.

Melanoma Cell-Intrinsic PD-1 Receptor Functions Promote Tumor Growth.

Therapeutic antibodies targeting programmed cell death 1 (PD-1) activate tumor-specific immunity and have shown remarkable efficacy in the treatment of melanoma. Yet, little is known about tumor cell-intrinsic PD-1 pathway effects. Here, we show that murine and human melanomas contain PD-1-expressing cancer subpopulations and demonstrate that melanoma cell-intrinsic PD-1 promotes tumorigenesis, even in mice lacking adaptive immunity. PD-1 inhibition on melanoma cells by RNAi, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised, and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, as does engagement of melanoma-PD-1 by its ligand, PD-L1, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuate growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor modulates downstream effectors of mTOR signaling. Our results identify melanoma cell-intrinsic functions of the PD-1:PD-L1 axis in tumor growth and suggest that blocking melanoma-PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy.

Ribosomal Proteins Regulate MHC Class I Peptide Generation for Immunosurveillance.

The MHC class I antigen presentation system enables T cell immunosurveillance of cancers and viruses. A substantial fraction of the immunopeptidome derives from rapidly degraded nascent polypeptides (DRiPs). By knocking down each of the 80 ribosomal proteins, we identified proteins that modulate peptide generation without altering source protein expression. We show that 60S ribosomal proteins L6 (RPL6) and RPL28, which are adjacent on the ribosome, play opposite roles in generating an influenza A virus-encoded peptide. Depleting RPL6 decreases ubiquitin-dependent peptide presentation, whereas depleting RPL28 increases ubiquitin-dependent and -independent peptide presentation. 40S ribosomal protein S28 (RPS28) knockdown increases total peptide supply in uninfected cells by increasing DRiP synthesis from non-canonical translation of "untranslated" regions and non-AUG start codons and sensitizes tumor cells for T cell targeting. Our findings raise the possibility of modulating immunosurveillance by pharmaceutical targeting ribosomes.

Sfp1 regulates transcriptional networks driving cell growth and division through multiple promoter-binding modes.

The yeast Sfp1 protein regulates both cell division and growth but how it coordinates these processes is poorly understood. We demonstrate that Sfp1 directly controls genes required for ribosome production and many other growth-promoting processes. Remarkably, the complete set of Sfp1 target genes is revealed only by a combination of ChIP (chromatin immunoprecipitation) and ChEC (chromatin endogenous cleavage) methods, which uncover two promoter binding modes, one requiring a cofactor and the other a DNA-recognition motif. Glucose-regulated Sfp1 binding at cell cycle "START" genes suggests that Sfp1 controls cell size by coordinating expression of genes implicated in mass accumulation and cell division.

RNA-binding motif protein 10 inactivates c-Myc by partnering with ribosomal proteins uL18 and uL5.

RNA-binding motif protein 10 (RBM10) is a frequently mutated tumor suppressor in lung adenocarcinoma (LUAD). Yet, it remains unknown whether cancer-derived mutant RBM10 compromises its tumor suppression function and, if so, the molecular insight of the underlying mechanisms. Here, we show that wild-type RBM10 suppresses lung cancer cell growth and proliferation by inactivating c-Myc that is essential for cancer cell survival. RBM10 directly binds to c-Myc and promotes c-Myc's ubiquitin-dependent degradation, while RBM10 knockdown leads to the induction of c-Myc level and activity. This negative action on c-Myc is further boosted by ribosomal proteins (RPs) uL18 (RPL5) and uL5 (RPL11) via their direct binding to RBM10. Cancer-derived mutant RBM10-I316F fails to bind to uL18 and uL5 and to inactivate c-Myc, thus incapable of suppressing tumorigenesis. Our findings uncover RBM10 as a pivotal c-Myc repressor by cooperating with uL18 and uL5 in lung cancer cells, as its failure to do so upon mutation favors tumorigenesis.

Cotton GhNAC4 promotes drought tolerance by regulating secondary cell wall biosynthesis and ribosomal protein homeostasis.

Drought has a severe impact on the quality and yield of cotton. Deciphering the key genes related to drought tolerance is important for understanding the regulation mechanism of drought stress and breeding drought-tolerant cotton cultivars. Several studies have demonstrated that NAC transcription factors are crucial in the regulation of drought stress, however, the related functional mechanisms are still largely unexplored. Here, we identified that NAC transcription factor GhNAC4 positively regulated drought stress tolerance in cotton. The expression of GhNAC4 was significantly induced by abiotic stress and plant hormones. Silencing of GhNAC4 distinctly impaired the resistance to drought stress and overexpressing GhNAC4 in cotton significantly enhanced the stress tolerance. RNA-seq analysis revealed that overexpression of GhNAC4 enriched the expression of genes associated with the biosynthesis of secondary cell walls and ribosomal proteins. We confirmed that GhNAC4 positively activated the expressions of GhNST1, a master regulator reported previously in secondary cell wall formation, and two ribosomal protein-encoding genes GhRPL12 and GhRPL18p, by directly binding to their promoter regions. Overexpression of GhNAC4 promoted the expression of downstream genes associated with the secondary wall biosynthesis, resulting in enhancing secondary wall deposition in the roots, and silencing of GhRPL12 and GhRPL18p significantly impaired the resistance to drought stress. Taken together, our study reveals a novel pathway mediated by GhNAC4 that promotes secondary cell wall biosynthesis to strengthen secondary wall development and regulates the expression of ribosomal protein-encoding genes to maintain translation stability, which ultimately enhances drought tolerance in cotton.

A Conserved Ribosomal Protein Has Entirely Dissimilar Structures in Different Organisms.

Ribosomes from different species can markedly differ in their composition by including dozens of ribosomal proteins that are unique to specific lineages but absent in others. However, it remains unknown how ribosomes acquire new proteins throughout evolution. Here, to help answer this question, we describe the evolution of the ribosomal protein msL1/msL2 that was recently found in ribosomes from the parasitic microorganism clade, microsporidia. We show that this protein has a conserved location in the ribosome but entirely dissimilar structures in different organisms: in each of the analyzed species, msL1/msL2 exhibits an altered secondary structure, an inverted orientation of the N-termini and C-termini on the ribosomal binding surface, and a completely transformed 3D fold. We then show that this fold switching is likely caused by changes in the ribosomal msL1/msL2-binding site, specifically, by variations in rRNA. These observations allow us to infer an evolutionary scenario in which a small, positively charged, de novo-born unfolded protein was first captured by rRNA to become part of the ribosome and subsequently underwent complete fold switching to optimize its binding to its evolving ribosomal binding site. Overall, our work provides a striking example of how a protein can switch its fold in the context of a complex biological assembly, while retaining its specificity for its molecular partner. This finding will help us better understand the origin and evolution of new protein components of complex molecular assemblies-thereby enhancing our ability to engineer biological molecules, identify protein homologs, and peer into the history of life on Earth.

The immunosuppressive drug cyclosporin A has an immunostimulatory function in CD8+ T cells.

Cyclosporin A is a well-established immunosuppressive drug used to treat or prevent graft-versus-host disease, the rejection of organ transplants, autoimmune disorders, and leukemia. It exerts its immunosuppressive effects by inhibiting calcineurin-mediated dephosphorylation of the nuclear factor of activated T cells (NFAT), thus preventing its nuclear entry and suppressing T cell activation. Here we report an unexpected immunostimulatory effect of cyclosporin A in activating the mammalian target of rapamycin complex 1 (mTORC1), a crucial metabolic hub required for T cell activation. Through screening a panel of tool compounds known to regulate mTORC1 activation, we found that cyclosporin A activated mTORC1 in CD8+ T cells in a 3-phosphoinositide-dependent protein kinase 1 (PDK1) and protein kinase B (PKB/AKT)-dependent manner. Mechanistically, cyclosporin A inhibited the calcineurin-mediated AKT dephosphorylation, thereby stabilizing mTORC1 signaling. Cyclosporin A synergized with mTORC1 pathway inhibitors, leading to potent suppression of proliferation and cytokine production in CD8+ T cells and an increase in the killing of acute T cell leukemia cells. Consequently, relying solely on CsA is insufficient to achieve optimal therapeutic outcomes. It is necessary to simultaneously target both the calcineurin-NFAT pathway and the mTORC1 pathway to maximize therapeutic efficacy.

The mitochondrial ribosomal protein mRpL4 regulates Notch signaling.

Mitochondrial ribosomal proteins (MRPs) assemble as specialized ribosome to synthesize mtDNA-encoded proteins, which are essential for mitochondrial bioenergetic and metabolic processes. MRPs are required for fundamental cellular activities during animal development, but their roles beyond mitochondrial protein translation are poorly understood. Here, we report a conserved role of the mitochondrial ribosomal protein L4 (mRpL4) in Notch signaling. Genetic analyses demonstrate that mRpL4 is required in the Notch signal-receiving cells to permit target gene transcription during Drosophila wing development. We find that mRpL4 physically and genetically interacts with the WD40 repeat protein wap and activates the transcription of Notch signaling targets. We show that human mRpL4 is capable of replacing fly mRpL4 during wing development. Furthermore, knockout of mRpL4 in zebrafish leads to downregulated expression of Notch signaling components. Thus, we have discovered a previously unknown function of mRpL4 during animal development.

Ribosome Collision Is Critical for Quality Control during No-Go Decay.

No-go decay (NGD) is a eukaryotic quality control mechanism that evolved to cope with translational arrests. The process is characterized by an endonucleolytic cleavage near the stall sequence, but the mechanistic details are unclear. Our analysis of cleavage sites indicates that cleavage requires multiple ribosomes on the mRNA. We also show that reporters harboring stall sequences near the initiation codon, which cannot accommodate multiple ribosomes, are not subject to NGD. Consistent with our model, we uncover an inverse correlation between ribosome density per mRNA and cleavage efficiency. Furthermore, promoting global ribosome collision in vivo resulted in ubiquitination of ribosomal proteins, suggesting that collision is sensed by the cell to initiate downstream quality control processes. Collectively, our data suggest that NGD and subsequent quality control are triggered by ribosome collision. This model provides insight into the regulation of quality control processes and the manner by which they reduce off-target effects.

Systems Phytohormone Responses to Mitochondrial Proteotoxic Stress.

Mitochondrial function is controlled by two separate genomes. This feature makes mitochondria prone to proteotoxic stress when a stoichiometric imbalance occurs in the protein complexes that perform oxidative phosphorylation, which consist of both nuclear- and mitochondrial-encoded proteins. Such a proteotoxic stress is known to induce the mitochondrial unfolded protein response (UPRmt) in animals. It is unknown whether UPRmt occurs in plants. Here, we induced a mitonuclear protein imbalance in Arabidopsis through chemical or genetic interference. Mitochondrial proteotoxic stress activated a plant-specific UPRmt and impaired plant growth and development. The plant UPRmt pathway is triggered by a transient oxidative burst, activating MAPK and hormonal (involving ethylene and auxin) signaling, which are all geared to repair proteostasis. This also establishes phytohormones as bona fide plant mitokines. Our data ascertain that mitochondrial protein quality control pathways, such as the UPRmt, are conserved in plants and that hormone signaling is an essential mediator that regulates mitochondrial proteostasis.

Engineering and functional analysis of yeast with a monotypic 40S ribosome subunit.

Emerging evidence reveals that ribosomes are not monolithic but dynamic machines with heterogeneous protein compositions that can reshape ribosomal translational abilities and cellular adaptation to environmental changes. Duplications of ribosomal protein (RP) genes are ubiquitous among organisms and are believed to affect cell function through paralog-specific regulation (e.g., by generating heterogeneous ribosomes) and/or gene dose amplification. However, direct evaluations of their impacts on cell function remain elusive due to the highly heterogeneous cellular RP pool. Here, we engineered a yeast with homogeneous 40S RP paralog compositions, designated homo-40S, by deleting the entire set of alternative duplicated genes encoding yeast 40S RP paralogs. Homo-40S displayed mild growth defects along with high sensitivity to the translation inhibitor paromomycin and a significantly increased stop codon readthrough. Moreover, doubling of the remaining RP paralogous genes in homo-40S rescued these phenotypes markedly, although not fully, compared to the wild-type phenotype, indicating that the dose of 40S RP genes together with the heterogeneity of the contents was vital for maintaining normal translational functionalities and growth robustness. Additional experiments revealed that homo-40S improved paromomycin tolerance via acquisition of bypass mutations or evolved to be diploid to generate fast-growing derivatives, highlighting the mutational robustness of engineered yeast to accommodate environmental and genetic changes. In summary, our work demonstrated that duplicated RP paralogs impart robustness and phenotypic plasticity through both gene dose amplification and paralog-specific regulation, paving the way for the direct study of ribosome biology through monotypic ribosomes with a homogeneous composition of specific RP paralogs.

Enolase of Streptococcus suis serotype 2 promotes biomolecular condensation of ribosomal protein SA for HBMECs apoptosis.

BACKGROUND: Ribosomal protein SA (RPSA) of human brain microvascular endothelial cells (HBMECs) can transfer from the cytosol to the cell surface and act as a receptor for some pathogens, including Streptococcus suis serotype 2 (SS2), a zoonotic pathogen causing meningitis in pigs and humans. We previously reported that SS2 virulence factor enolase (ENO) binds to RPSA on the cell surface of HBMECs and induces apoptosis. However, the mechanism that activates RPSA translocation to the cell surface and induces ENO-mediated HBMEC apoptosis is unclear. RESULTS: Here, we show that RPSA localization and condensation on the host cell surface depend on its internally disordered region (IDR). ENO binds to the IDR of RPSA and promotes its interaction with RPSA and vimentin (VIM), which is significantly suppressed after 1,6-Hexanediol (1,6-Hex, a widely used tool to disrupt phase separation) treatment, indicating that ENO incorporation and thus the concentration of RPSA/VIM complexes via co-condensation. Furthermore, increasing intracellular calcium ions (Ca2+) in response to SS2 infection further facilitates the liquid-like condensation of RPSA and aggravates ENO-induced HBMEC cell apoptosis. CONCLUSIONS: Together, our study provides a previously underappreciated molecular mechanism illuminating that ENO-induced RPSA condensation activates the migration of RPSA to the bacterial cell surface and stimulates SS2-infected HBMEC death and, potentially, disease progression. This study offers a fresh avenue for investigation into the mechanism by which other harmful bacteria infect hosts via cell surfaces' RPSA.

Ribosomal-processing cysteine protease homolog modulates Streptococcus mutans glucan production and interkingdom interactions.

Glucan-dependent biofilm formation is a crucial process in the establishment of Streptococcus mutans as a cariogenic oral microbe. The process of glucan formation has been investigated in great detail, with glycosyltransferases GtfB, GtfC, and GtfD shown to be indispensable for the synthesis of glucans from sucrose. Glucan production can be visualized during biofilm formation through fluorescent labeling, and its abundance, as well as the effect of glucans on general biofilm architecture, is a common phenotype to study S. mutans virulence regulation. Here, we describe an entirely new phenotype associated with glucan production, caused by a mutation in the open reading frame SMU_848, which is located in an operon encoding ribosome-associated proteins. This mutation led to the excess production and accumulation of glucan-containing droplets on the surface of biofilms formed on agar plates after prolonged incubation. While not characterized in S. mutans, SMU_848 shows homology to the phage-related ribosomal protease Prp, essential in cleaving off the N-terminal extension of ribosomal protein L27 for functional ribosome assembly in Staphylococcus aureus. We present a further characterization of SMU_848/Prp, demonstrating that the deletion of this gene leads to significant changes in S. mutans gtfBC expression. Surprisingly, it also profoundly impacts the interkingdom interaction between S. mutans and Candida albicans, a relevant dual-species interaction implicated in severe early childhood caries. The presented data support a potential broader role for SMU_848/Prp, possibly extending its functionality beyond the ribosomal network to influence important ecological processes. IMPORTANCE: Streptococcus mutans is an important member of the oral biofilm and is implicated in the initiation of caries. One of the main virulence mechanisms is the glucan-dependent formation of biofilms. We identified a new player in the regulation of glucan production, SMU_848, which is part of an operon that also encodes for ribosomal proteins L27 and L21. A mutation in SMU_848, which encodes a phage-related ribosomal protease Prp, leads to a significant accumulation of glucan-containing droplets on S. mutans biofilms, a previously unknown phenotype. Further investigations expanded our knowledge about the role of SMU_848 beyond its role in glucan production, including significant involvement in interkingdom interactions, thus potentially playing a global role in the virulence regulation of S. mutans.

In vivo evidence for homeostatic regulation of ribosomal protein levels in Drosophila.

The ribosome is a molecular machine essential for protein synthesis, which is composed of approximately 80 different ribosomal proteins (Rps). Studies in yeast and cell culture systems have revealed that the intracellular level of Rps is finely regulated by negative feedback mechanisms or ubiquitin-proteasome system, which prevents over- or under-abundance of Rps in the cell. However, in vivo evidence for the homeostatic regulation of intracellular Rp levels has been poor. Here, using Drosophila genetics, we show that intracellular Rp levels are regulated by proteasomal degradation of excess Rps that are not incorporated into the ribosome. By establishing an EGFP-fused Rp gene system that can monitor endogenously expressed Rp levels, we found that endogenously expressed EGFP-RpS20 or -RpL5 is eliminated from the cell when RpS20 or RpL5 is exogenously expressed. Notably, the level of endogenously expressed Hsp83, a housekeeping gene, was not affected by exogenous expression of Hsp83, suggesting that the strict negative regulation of excess protein is specific for intracellular Rps. Further analyses revealed that the maintenance of cellular Rp levels is not regulated at the transcriptional level but by proteasomal degradation of excess free Rps as a protein quality control mechanism. Our observations provide not only the in vivo evidence for the homeostatic regulation of Rp levels but also a novel genetic strategy to study in vivo regulation of intracellular Rp levels and its role in tissue homeostasis via cell competition.Key words: ribosomal protein, proteasomal degradation, Drosophila.

Ribosomal protein RPL11 haploinsufficiency causes anemia in mice via activation of the RP-MDM2-p53 pathway.

Recent discovery of the ribosomal protein (RP) RPL11 interacting with and inhibiting the E3 ubiquitin ligase function of MDM2 established the RP-MDM2-p53 signaling pathway, which is linked to biological events, including ribosomal biogenesis, nutrient availability, and metabolic homeostasis. Mutations in RPs lead to a diverse array of phenotypes known as ribosomopathies in which the role of p53 is implicated. Here, we generated conditional RPL11-deletion mice to investigate in vivo effects of impaired RP expression and its functional connection with p53. While deletion of one Rpl11 allele in germ cells results in embryonic lethality, deletion of one Rpl11 allele in adult mice does not affect viability but leads to acute anemia. Mechanistically, we found RPL11 haploinsufficiency activates p53 in hematopoietic tissues and impedes erythroid precursor differentiation, resulting in insufficient red blood cell development. We demonstrated that reducing p53 dosage by deleting one p53 allele rescues RPL11 haploinsufficiency-induced inhibition of erythropoietic precursor differentiation and restores normal red blood cell levels in mice. Furthermore, blocking the RP-MDM2-p53 pathway by introducing an RP-binding mutation in MDM2 prevents RPL11 haploinsufficiency-caused p53 activation and rescues the anemia in mice. Together, these findings demonstrate that the RP-MDM2-p53 pathway is a critical checkpoint for RP homeostasis and that p53-dependent cell cycle arrest of erythroid precursors is the molecular basis for the anemia phenotype commonly associated with RP deficiency.

The Neurobeachin-like 2 protein (NBEAL2) controls the homeostatic level of the ribosomal protein RPS6 in mast cells.

The Beige and Chediak-Higashi (BEACH) domain-containing, Neurobeachin-like 2 (NBEAL2) protein is a molecule with a molecular weight of 300 kDa. Inactivation of NBEAL2 by loss-of-function mutations in humans as well as deletion of the Nbeal2 gene in mice results in functional defects in cells of the innate immune system such as neutrophils, NK-cells, megakaryocytes, platelets and of mast cells (MCs). To investigate the detailed function of NBEAL2 in murine MCs we generated MCs from wild type (wt) and Nbeal2-/- mice, and deleted Nbeal2 by CRISPR/Cas9 technology in the murine mast cell line MC/9. We also predicted the structure of NBEAL2 to infer its function and to examine potential mechanisms for its association with interaction partners by using the deep learning-based method RoseTTAFold and the Pymol© software. The function of NBEAL2 was analysed by molecular and immunological techniques such as co-immunoprecipitation (co-IP) experiments, western blotting, enzyme-linked immunosorbent assay and flow cytometry. We identified RPS6 as an interaction partner of NBEAL2. Thereby, the NBEAL2/RPS6 complex formation is probably required to control the protein homeostasis of RPS6 in MCs. Consequently, inactivation of NBEAL2 leads to accumulation of strongly p90RSK-phosphorylated RPS6 molecules which results in the development of an abnormal MC phenotype characterised by prolonged growth factor-independent survival and in a pro-inflammatory MC-phenotype.

Suppressor analysis links trans-translation and ribosomal protein uS7 to RluD function in Escherichia coli.

The pseudouridine (ψ) synthase, RluD is responsible for three ψ modifications in the helix 69 (H69) of bacterial 23S rRNA. While normally dispensable, rluD becomes critical for rapid cell growth in bacteria that are defective in translation-termination. In slow-growing rluD- bacteria, suppressors affecting termination factors RF2 and RF3 arise frequently and restore normal termination and rapid cell growth. Here we describe two weaker suppressors, affecting rpsG, encoding ribosomal protein uS7 and ssrA, encoding tmRNA. In K-12 strains of E. coli, rpsG terminates at a TGA codon. In the suppressor strain, alteration of an upstream CAG to a TAG stop codon results in a shortened uS7 and partial alleviation of slow growth, likely by replacing an inefficient TGA stop codon with the more efficient TAG. Inefficient termination events, such as occurs in some rluD- strains, are targeted by trans-translation. Inactivation of the ssrA gene in slow-growing, termination-defective mutants lacking RluD and RF3, also partially restores robust growth, most probably by preventing destruction of completed polypeptides on ribosomes at slow-terminating stop codons. Finally, an additional role for RluD has been proposed, independent of its pseudouridine synthase activity. This is based on the observation that plasmids expressing catalytically dead (D139N or D139T) RluD proteins could nonetheless restore robust growth to an E. coli K-12 rluD- mutant. However, newly constructed D139N and D139T rluD plasmids do not have any growth-restoring activity and the original observations were likely due to the appearance of suppressors.

DNA methylation-mediated epigenetic regulation of oncogenic RPS2 as a novel therapeutic target and biomarker in hepatocellular carcinoma.

Ribosomal Protein S2 (RPS2) has emerged as a potential prognostic biomarker due to its involvement in key cellular processes and its altered expression pattern in certain types of cancer. However, its role in hepatocellular carcinoma (HCC) has yet to be investigated. Herein, we analyzed RPS2 mRNA expression and promoter methylation in HCC patient samples and HepG2 cells. Subsequently, loss-of-function experiments were conducted to determine the function of RPS2 in HCC cells in vitro. Our results revealed that RPS2 mRNA expression is significantly elevated, and its promoter is hypomethylated in HCC patient samples compared to controls. In addition, 5-Azacytidine treatment in HepG2 cells decreased RPS2 promoter methylation level and increased its mRNA expression. RPS2 knockdown in HepG2 cells suppressed cell proliferation and promoted apoptosis. Functional pathway analysis of genes positively and negatively associated with RPS2 expression in HCC showed enrichment in ribosomal biogenesis, translation machinery, cell cycle regulation, and DNA processing. Furthermore, utilizing drug-protein 3D docking, we found that doxorubicin, sorafenib, and 5-Fluorouracil, showed high affinity to the active sites of RPS2, and in vitro treatment with these drugs reduced RPS2 expression. For the first time, we report on DNA methylation-mediated epigenetic regulation of RPS2 and its oncogenic role in HCC. Our findings suggest that RPS2 plays a significant role in the development and progression of HCC, hence its potential prognostic and therapeutic utility. Moreover, as epigenetic changes happen early in cancer development, RPS2 may serve as a potential biomarker for tumor progression.