A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies.

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and in vivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how in vivo tissue-specific phenotypes emerge in ribosomopathies.

Ribosome Biogenesis and Cancer: Overview on Ribosomal Proteins.

Cytosolic ribosomes (cytoribosomes) are macromolecular ribonucleoprotein complexes that are assembled from ribosomal RNA and ribosomal proteins, which are essential for protein biosynthesis. Mitochondrial ribosomes (mitoribosomes) perform translation of the proteins essential for the oxidative phosphorylation system. The biogenesis of cytoribosomes and mitoribosomes includes ribosomal RNA processing, modification and binding to ribosomal proteins and is assisted by numerous biogenesis factors. This is a major energy-consuming process in the cell and, therefore, is highly coordinated and sensitive to several cellular stressors. In mitochondria, the regulation of mitoribosome biogenesis is essential for cellular respiration, a process linked to cell growth and proliferation. This review briefly overviews the key stages of cytosolic and mitochondrial ribosome biogenesis; summarizes the main steps of ribosome biogenesis alterations occurring during tumorigenesis, highlighting the changes in the expression level of cytosolic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) in different types of tumors; focuses on the currently available information regarding the extra-ribosomal functions of CRPs and MRPs correlated to cancer; and discusses the role of CRPs and MRPs as biomarkers and/or molecular targets in cancer treatment.

A functional connection between translation elongation and protein folding at the ribosome exit tunnel in Saccharomyces cerevisiae.

Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5' region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel.

How Altered Ribosome Production Can Cause or Contribute to Human Disease: The Spectrum of Ribosomopathies.

A number of different defects in the process of ribosome production can lead to a diversified spectrum of disorders that are collectively identified as ribosomopathies. The specific factors involved may either play a role only in ribosome biogenesis or have additional extra-ribosomal functions, making it difficult to ascribe the pathogenesis of the disease specifically to an altered ribosome biogenesis, even if the latter is clearly affected. We reviewed the available literature in the field from this point of view with the aim of distinguishing, among ribosomopathies, the ones due to specific alterations in the process of ribosome production from those characterized by a multifactorial pathogenesis.

Ribosomal protein S3 selectively affects colon cancer growth by modulating the levels of p53 and lactate dehydrogenase.

Ribosomal protein S3 (RPS3) is a component of the 40S ribosomal subunit. It is known to function in ribosome biogenesis and as an endonuclease. RPS3 has been shown to be over expressed in colon adenocarcinoma but its role in colon cancer is still unknown. In this study, we aim at determining the expression levels of RPS3 in a colon cancer cell line Caco-2 compared to a normal colon mucosa cell line NCM-460 and study the effects of targeting this protein by siRNA on cellular behavior. RPS3 was found to be expressed in both cell lines. However, siRNA treatment showed a more protruding effect on Caco-2 cells compared to NCM-460 cells. RPS3 knockdown led to a significant decrease in the proliferation, survival, migration and invasion and an increase in the apoptosis of Caco-2 cells. Western blot analysis demonstrated that these effects correlated with an increase in the level of the tumor suppressor p53 and a decrease in the level and activity of lactate dehydrogenase (LDH), an enzyme involved in the metabolism of cancer cells. No significant effect was shown in normal colon NCM-460 cells. Targeting p53 by siRNA did not affect RPS3 levels indicating that p53 may be a downstream target of RPS3. However, the concurrent knockdown of RPS3 and p53 showed no change in LDH level in Caco-2 cells suggesting an interesting interplay among the three proteins. These findings might present RPS3 as a selective molecular marker in colon cancer and an attractive target for colon cancer therapy.

Extra-ribosomal functions of Mtb RpsB in imparting stress resilience and drug tolerance to mycobacteria.

Emerging observations suggest that ribosomal proteins (RPs) play important extra-ribosomal roles in maintenance of cellular homeostasis. However, the mechanistic insights into these processes have not been extensively explored, especially in pathogenic bacteria. Here, we present our findings on potential extra-ribosomal functions of Mycobacterium tuberculosis (Mtb) RPs. We observed that Mtb RpsB and RpsQ are differentially localized to cell wall fraction in M. tuberculosis (H37Rv), while their M. smegmatis (Msm) homologs are primarily cytosolic. Cellular fractionation of ectopically expressed Mtb RPs in surrogate host (M. smegmatis) also shows their association with cell membrane/cell wall without any gross changes in cell morphology. M. smegmatis expressing Mtb RpsB exhibited altered redox homeostasis, decreased drug-induced ROS, reduced cell wall permeability and increased tolerance to various proteotoxic stress (oxidative stress, SDS and starvation). Mtb RpsB expression was also associated with increased resistance specifically towards Isoniazid, Ethionamide and Streptomycin. The enhanced drug tolerance was specific to Mtb RpsB and not observed upon ectopic expression of M. smegmatis homolog (Msm RpsB). Interestingly, C-terminus deletion in Mtb RpsB affected its localization and reversed the stress-resilient phenotypes. We also observed that M. tuberculosis (H37Rv) with upregulated RpsB levels had higher intracellular survival in macrophage. All these observations hint towards existence of moonlighting roles of Mtb RpsB in imparting stress resilience to mycobacteria. This work open avenues for further exploration of alternative pathways associated with fitness and drug tolerance in mycobacteria.

Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) was the first ribosomopathy described and is a constitutional inherited bone marrow failure syndrome. Erythroblastopenia is the major characteristic of the disease, which is a model for ribosomal diseases, related to a heterozygous allelic variation in 1 of the 20 ribosomal protein genes of either the small or large ribosomal subunit. The salient feature of classical DBA is a defect in ribosomal RNA maturation that generates nucleolar stress, leading to stabilization of p53 and activation of its targets, resulting in cell-cycle arrest and apoptosis. Although activation of p53 may not explain all aspects of DBA erythroid tropism, involvement of GATA1/HSP70 and globin/heme imbalance, with an excess of the toxic free heme leading to reactive oxygen species production, account for defective erythropoiesis in DBA. Despite significant progress in defining the molecular basis of DBA and increased understanding of the mechanistic basis for DBA pathophysiology, progress in developing new therapeutic options has been limited. However, recent advances in gene therapy, better outcomes with stem cell transplantation, and discoveries of putative new drugs through systematic drug screening using large chemical libraries provide hope for improvement.

Overexpressed circ-RPL15 predicts poor survival and promotes the progression of gastric cancer via regulating miR-502-3p/OLFM4/STAT3 pathway.

BACKGROUND: Increasing studies have revealed that circular RNAs (circRNAs) contribute to gastric cancer (GC) progression. The circular RNA ribosomal protein L15 (circ-RPL15) is involved in chronic lymphocytic leukemia. However, its expression and functions in GC remain elusive. METHODS: The expression of circ-RPL15 in human GC tissues and adjacent normal tissues, human gastric cancer cell lines (MGC-803, BGC-823, MGN-28, SGC-7901, AGS) and normal gastric mucosal epithelial cell line (GES-1) were detected by RT-PCR. The relationship between circ-RPL15 level and clinical-pathological indicators were also analyzed. Gain- of function experiments of circ-RPL15 and miR-502-3p were conducted to verify their roles in mediating GC cell proliferation, apoptosis and metastasis. Also, the downstream mechanisms of circ-RPL15 were predicted by bioinformatics analysis, and the interactions between circ-RPL15 and miR-502-3p, miR-502-3p and OLFM4 were verified by dual luciferase reporter gene assay and RNA FISH. RESULTS: circ-RPL15 was upregulated in GC tissues and cell lines, and the overexpressed circ-RPL15 was correlated with poorer survival of GC patients. Functionally, circ-RPL15 upregulation distinctly promoted the proliferation, migration and invasion of GC cells and inhibited apoptosis. Mechanistically, circ-RPL15 functioned as a competitive endogenous RNA via sponging miR-502-3p and activated OLFM4/STAT3 pathway. CONCLUSION: circ-RPL15 promotes GC progression and predicts poor prognosis of GC patients, and regulates the malignant phenotypes of GC cells by mediating the miR-502-3p/OLFM4/STAT3 axis.

Post-transcriptional regulation of C-C motif chemokine ligand 2 expression by ribosomal protein L22 during LPS-mediated inflammation.

Monocyte infiltration to the site of pathogenic invasion is critical for inflammatory response and host defence. However, this process demands precise regulation as uncontrolled migration of monocytes to the site delays resolution of inflammation and ultimately promotes chronic inflammation. C-C motif chemokine ligand 2 (CCL2) plays a key role in monocyte migration, and hence, its expression should be tightly regulated. Here, we report a post-transcriptional regulation of CCL2 involving the large ribosomal subunit protein L22 (RPL22) in LPS-activated, differentiated THP-1 cells. Early events following LPS treatment include transcriptional upregulation of RPL22 and its nuclear accumulation. The protein binds to the first 20 nt sequence of the 5'UTR of ccl2 mRNA. Simultaneous nuclear translocation of up-frameshift-1 protein and its interaction with RPL22 results in cytoplasmic degradation of the ccl2 mRNA at a later stage. Removal of RPL22 from cells results in increased expression of CCL2 in response to LPS causing disproportionate migration of monocytes. We propose that post-transcriptional regulation of CCL2 by RPL22 fine-tunes monocyte infiltration during a pathogenic insult and maintains homeostasis of the immune response critical to resolution of inflammation. DATABASES: Microarray data are available in NCBI GEO database (Accession No GSE126525).

RPS7 promotes cell migration through targeting epithelial-mesenchymal transition in prostate cancer.

OBJECTIVES: Small ribosomal protein subunit 7 (RPS7) is an important structural components of the ribosome involved in protein synthesis, previous studies demonstrated that RPS7 was associated with several malignancies, but the role of RPS7 in prostate cancer (PCa) remains unclear. To decipher such a puzzle, in the current study, we deciphered the role and mechanism of RPS7 during the progression of PCa. MATERIAL AND METHODS: In this study, the expression of mRNA was performed by quantitative real-time PCR. The protein level was identified by Western blotting. Kaplan-Meier survival analysis was demonstrated the relation between the abnormal expression of RPS7 mRNA and the overall survival. Cell proliferation was assessed by MTT assay and cell counting, meanwhile, cell migration was checked by transwell assay. RESULTS: RPS7 is higher expressed in PCa (p < 0.001), and the overexpression of RPS7 is closely associated with poor outcome of PCa patients after radical prostatectomy (p < 0.001). Inhibition the expression of RPS7 with a specific RPS7 siRNA could markedly attenuate prostate tumor growth and migration (p < 0.05). Mechanistic data reveals that inhibition of RPS7 could up-regulate the epithelial protein marker, E-cadherin (p < 0.05), and down-regulate the mesenchymal protein markers, such as N-cadherin and Snail (p < 0.001). CONCLUSIONS: RPS7 is a newly verified tumor promoter in PCa, and promotes cell migration by targeting epithelial-to-mesenchymal transitionpathway. Thus, inhibition of RPS7-epithelial to-mesenchymal transition signaling might represent a prospective approach toward limiting prostate tumor progression.

Rps14, Csnk1a1 and miRNA145/miRNA146a deficiency cooperate in the clinical phenotype and activation of the innate immune system in the 5q- syndrome.

RPS14, CSNK1A1, and miR-145 are universally co-deleted in the 5q- syndrome, but mouse models of each gene deficiency recapitulate only a subset of the composite clinical features. We analyzed the combinatorial effect of haploinsufficiency for Rps14, Csnk1a1, and miRNA-145, using mice with genetically engineered, conditional heterozygous inactivation of Rps14 and Csnk1a1 and stable knockdown of miR-145/miR-146a. Combined Rps14/Csnk1a1/miR-145/146a deficiency recapitulated the cardinal features of the 5q- syndrome, including (1) more severe anemia with faster kinetics than Rps14 haploinsufficiency alone and (2) pathognomonic megakaryocyte morphology. Macrophages, regulatory cells of erythropoiesis and the innate immune response, were significantly increased in Rps14/Csnk1a1/miR-145/146a deficient mice as well as in 5q- syndrome patient bone marrows and showed activation of the innate immune response, reflected by increased expression of S100A8, and decreased phagocytic function. We demonstrate that Rps14/Csnk1a1/miR-145 and miR-146a deficient macrophages alter the microenvironment and induce S100A8 expression in the mesenchymal stem cell niche. The increased S100A8 expression in the mesenchymal niche was confirmed in 5q- syndrome patients. These data indicate that intrinsic defects of the 5q- syndrome hematopoietic stem cell directly alter the surrounding microenvironment, which in turn affects hematopoiesis as an extrinsic mechanism.

Kinase PIM1 promotes prostate cancer cell growth via c-Myc-RPS7-driven ribosomal stress.

Ribosomal stress is known to increase cancer risk; however, the molecular mechanism underlying its various effects on cancer remains unclear. To decipher this puzzle, we investigated the upstream signaling pathway that might be involved in promoting ribosomal stress that leads to tumor progression. Our results suggested that inhibition of kinase PIM1 attenuated PC3 cell growth and motility following the condensed cellular body and decreased protein translation in PIM1-inhibited cells. In addition, PIM1 was found to be a component of the small 40S ribosomal subunit and could regulate the expression of ribosomal small subunit protein 7 (RPS7). Our investigation also revealed that PIM1 enhanced the protein stability of c-Myc. Furthermore, a functional E-box motif was found upstream of the transcription start site in RPS7, and RPS7 has been proven to be a transcriptional target of c-Myc. Additionally, knocking down RPS7 dramatically reduced cell growth in vitro and in vivo, whereas enhancing RPS7 expression reversed the condensed cellular body and decreased protein translation resulted from PIM1 inhibition. Finally, biochemical recurrence-free survival and overall survival analysis indicated that the concomitant upregulation of PIM1 and RPS7 correlated with the worst prognosis of prostate cancer (PCa). Overall, our results demonstrated that kinase PIM1 promotes cell growth through c-Myc-RPS7-induced ribosomal stress in PCa. These findings substantially expanded our understanding on the molecular mechanism of PIM1-promoted abnormal ribosomal biosynthesis in tumorigenesis and tumor progression in PCa. Therapies that target molecules involved in PIM1-RPS7-induced ribosomal stress could provide a promising approach to treating PCa.

Ribosomal protein L14 contributes to the early assembly of 60S ribosomal subunits in Saccharomyces cerevisiae.

The contribution of most ribosomal proteins to ribosome synthesis has been quite well analysed in Saccharomyces cerevisiae. However, few yeast ribosomal proteins still await characterization. Herein, we show that L14, an essential 60S ribosomal protein, assembles in the nucleolus at an early stage into pre-60S particles. Depletion of L14 results in a deficit in 60S subunits and defective processing of 27SA2 and 27SA3 to 27SB pre-rRNAs. As a result, 27S pre-rRNAs are subjected to turnover and export of pre-60S particles is blocked. These phenotypes likely appear as the direct consequence of the reduced pre-60S particle association not only of L14 upon its depletion but also of a set of neighboring ribosomal proteins located at the solvent interface of 60S subunits and the adjacent region surrounding the polypeptide exit tunnel. These pre-60S intermediates also lack some essential trans-acting factors required for 27SB pre-rRNA processing but accumulate practically all factors required for processing of 27SA3 pre-rRNA. We have also analysed the functional interaction between the eukaryote-specific carboxy-terminal extensions of the neighboring L14 and L16 proteins. Our results indicate that removal of the most distal parts of these extensions cause slight translation alterations in mature 60S subunits.

Involvement of RPL11 in the enhancement of P53 stability by a podophyllum derivative, a topoisomerase II inhibitor.

In previous work, we presented experimental and theoretical evidence that D-3F or 4-N-(2-Amino-3-fluoropyridine)-4-deoxidation-4'-demethylepipofophyllotoxin induced G2 /M phase arrest and apoptosis, purportedly by increasing the expression of P53. However, the precise mechanism of D-3F action is currently unknown. Here, we investigated the mechanism by which D-3F treatment induces increased expression of P53. This study showed that D-3F definitively inhibited the activity of topoisomerase II in a dose-dependent manner and resulted in DNA damage. The results were in overall agreement with modeling and docking studies performed on D-3F. In addition, D-3F increased the levels of P53 and P21 in HeLa cells in a dose-dependent manner, this in turn prolonged the half-life of P53. Taken together, these data suggested that D-3F-mediated transient enhancement of P53 stabilization may be critical for the P53/P21 signalling pathway leading to G2 /M phase arrest on HeLa cells. Furthermore, D-3F downregulated the phosphorylation of E3 ubiquitin-protein ligase murine double minute 2 (Mdm2) at Ser166, inhibited Mdm2-mediated ubiquitination of P53, and released 60S ribosomal protein L11 (RPL11) from the nucleolus into the nucleoplasm. To conclude, the topoisomerase II inhibitor D-3F causes P53 to accumulate in HeLa cell lines by enhancing its stability as a result of DNA-damage induced RPL11 relocalization and subsequent blocking of the P53-Mdm2 feedback loop.

Rpl22 Loss Selectively Impairs αß T Cell Development by Dysregulating Endoplasmic Reticulum Stress Signaling.

Although ribosomal proteins (RP) are thought to primarily facilitate biogenesis of the ribosome and its ability to synthesize protein, emerging evidence suggests that individual RP can perform critical regulatory functions that control developmental processes. We showed previously that despite the ubiquitous expression of the RP ribosomal protein L22 (Rpl22), germline ablation of Rpl22 in mice causes a selective, p53-dependent block in the development of αß, but not γδ, T cell progenitors. Nevertheless, the basis by which Rpl22 loss selectively induces p53 in αß T cell progenitors remained unclear. We show in this study that Rpl22 regulates the development of αß T cells by restraining endoplasmic reticulum (ER) stress responses. In the absence of Rpl22, ER stress is exacerbated in αß, but not γδ, T cell progenitors. The exacerbated ER stress in Rpl22-deficient αß T lineage progenitors is responsible for selective induction of p53 and their arrest, as pharmacological induction of stress is sufficient to induce p53 and replicate the selective block of αß T cells, and attenuation of ER stress signaling by knockdown of protein kinase R-like ER kinase, an ER stress sensor, blunts p53 induction and rescues development of Rpl22-deficient αß T cell progenitors. Rpl22 deficiency appears to exacerbate ER stress by interfering with the ability of ER stress signals to block new protein synthesis. Our finding that Rpl22 deficiency exacerbates ER stress responses and induces p53 in αß T cell progenitors provides insight into how a ubiquitously expressed RP can perform regulatory functions that are selectively required by some cell lineages but not others.

The role of ribosomal proteins in the regulation of cell proliferation, tumorigenesis, and genomic integrity.

Ribosomal proteins (RPs), the essential components of the ribosome, are a family of RNA-binding proteins, which play prime roles in ribosome biogenesis and protein translation. Recent studies revealed that RPs have additional extra-ribosomal functions, independent of protein biosynthesis, in regulation of diverse cellular processes. Here, we review recent advances in our understanding of how RPs regulate apoptosis, cell cycle arrest, cell proliferation, neoplastic transformation, cell migration and invasion, and tumorigenesis through both MDM2/p53-dependent and p53-independent mechanisms. We also discuss the roles of RPs in the maintenance of genome integrity via modulating DNA damage response and repair. We further discuss mutations or deletions at the somatic or germline levels of some RPs in human cancers as well as in patients of Diamond-Blackfan anemia and 5q- syndrome with high susceptibility to cancer development. Moreover, we discuss the potential clinical application, based upon abnormal levels of RPs, in biomarker development for early diagnosis and/or prognosis of certain human cancers. Finally, we discuss the pressing issues in the field as future perspectives for better understanding the roles of RPs in human cancers to eventually benefit human health.

Ribosomal Protein Rpl22 Controls the Dissemination of T-cell Lymphoma.

Mutations in ribosomal proteins cause bone marrow failure syndromes associated with increased cancer risk, but the basis by which they do so remains unclear. We reported previously that the ribosomal protein Rpl22 is a tumor suppressor in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), and that loss of just one Rpl22 allele accelerates T-cell lymphomagenesis by activating NF-κB and inducing the stem cell factor Lin28B. Here, we show that, paradoxically, loss of both alleles of Rpl22 restricts lymphoma progression through a distinct effect on migration of malignant cells out of the thymus. Lymphoma-prone AKT2-transgenic or PTEN-deficient mice on an Rpl22(-/-) background developed significantly larger and markedly more vascularized thymic tumors than those observed in Rpl22(+/+) control mice. But, unlike Rpl22(+/+) or Rpl22(+/-) tumors, Rpl22(-/-) lymphomas did not disseminate to the periphery and were retained in the thymus. We traced the defect in the Rpl22(-/-) lymphoma migratory capacity to downregulation of the KLF2 transcription factor and its targets, including the key migratory factor sphingosine 1-phosphate receptor 1 (S1PR1). Indeed, reexpression of S1PR1 in Rpl22-deficient tumor cells restores their migratory capacity in vitro The regulation of KLF2 and S1PR1 by Rpl22 appears to be proximal as Rpl22 reexpression in Rpl22-deficient lymphoma cells restores expression of KLF2 and S1P1R, while Rpl22 knockdown in Rpl22-sufficient lymphomas attenuates their expression. Collectively, these data reveal that, while loss of one copy of Rpl22 promotes lymphomagenesis and disseminated disease, loss of both copies impairs responsiveness to migratory cues and restricts malignant cells to the thymus. Cancer Res; 76(11); 3387-96. ©2016 AACR.

Hydrogen peroxide regulates cell adhesion through the redox sensor RPSA.

To become metastatic, a tumor cell must acquire new adhesion properties that allow migration into the surrounding connective tissue, transmigration across endothelial cells to reach the blood stream and, at the site of metastasis, adhesion to endothelial cells and transmigration to colonize a new tissue. Hydrogen peroxide (H2O2) is a redox signaling molecule produced in tumor cell microenvironment with high relevance for tumor development. However, the molecular mechanisms regulated by H2O2 in tumor cells are still poorly known. The identification of H2O2-target proteins in tumor cells and the understanding of their role in tumor cell adhesion are essential for the development of novel redox-based therapies for cancer. In this paper, we identified Ribosomal Protein SA (RPSA) as a target of H2O2 and showed that RPSA in the oxidized state accumulates in clusters that contain specific adhesion molecules. Furthermore, we showed that RPSA oxidation improves cell adhesion efficiency to laminin in vitro and promotes cell extravasation in vivo. Our results unravel a new mechanism for H2O2-dependent modulation of cell adhesion properties and identify RPSA as the H2O2 sensor in this process. This work indicates that high levels of RPSA expression might confer a selective advantage to tumor cells in an oxidative environment.

Regulatory role of rpL3 in cell response to nucleolar stress induced by Act D in tumor cells lacking functional p53.

Many chemotherapeutic drugs cause nucleolar stress and p53-independent pathways mediating the nucleolar stress response are emerging. Here, we demonstrate that ribosomal stress induced by Actinomycin D (Act D) is associated to the up-regulation of ribosomal protein L3 (rpL3) and its accumulation as ribosome-free form in lung and colon cancer cell lines devoid of p53. Free rpL3 regulates p21 expression at transcriptional and post-translational levels through a molecular mechanism involving extracellular-signal-regulated kinases1/2 (ERK1/2) and mouse double minute-2 homolog (MDM2). Our data reveal that rpL3 participates to cell response acting as a critical regulator of apoptosis and cell migration. It is noteworthy that silencing of rpL3 abolishes the cytotoxic effects of Act D suggesting that the loss of rpL3 makes chemotherapy drugs ineffective while rpL3 overexpression associates to a strong increase of Act D-mediated inhibition of cell migration. Taking together our results show that the efficacy of Act D chemotherapy depends on rpL3 status revealing new specific targets involved in the molecular pathways activated by Act D in cancers lacking of p53. Hence, the development of treatments aimed at upregulating rpL3 may be beneficial for the treatment of these cancers.

60S ribosomal protein L35 regulates ß-casein translational elongation and secretion in bovine mammary epithelial cells.

60S ribosomal protein L35 (RPL35) is an important component of the 60S ribosomal subunit and has a role in protein translation and endoplasmic reticulum (ER) docking. However, few studies have investigated RPL35 in eukaryotes and much remains to be learned. Here, we analyzed the function of RPL35 in ß-casein (CSN2) synthesis and secretion in bovine mammary epithelial cells (BMECs). We found that methionine (Met) could promote the expressions of CSN2 and RPL35. Analysis of overexpression and inhibition of RPL35 confirmed that it could mediate the Met signal and regulate CSN2 expression. The mechanism of CSN2 regulation by RPL35 was analyzed by coimmunoprecipitation (Co-IP), colocalization, fluorescence resonance energy transfer (FRET) and gene mutation. We found that RPL35 could control ribosome translational elongation during synthesis of CSN2 by interacting with eukaryotic translational elongation factor 2 (eEF2), and that eEF2 was the signaling molecule downstream of RPL35 controlling this process. RPL35 could also control the secretion of CSN2 by locating it to the ER. Taken together, these results revealed that, RPL35 was an important positive regulatory factor involving in the Met-mediated regulation of CSN2 translational elongation and secretion.