Mitochondrial Ribosomal Protein MRPS15 Is a Component of Cytosolic Ribosomes and Regulates Translation in Stressed Cardiomyocytes.

Regulation of mRNA translation is a crucial step in controlling gene expression in stressed cells, impacting many pathologies, including heart ischemia. In recent years, ribosome heterogeneity has emerged as a key control mechanism driving the translation of subsets of mRNAs. In this study, we investigated variations in ribosome composition in human cardiomyocytes subjected to endoplasmic reticulum stress induced by tunicamycin treatment. Our findings demonstrate that this stress inhibits global translation in cardiomyocytes while activating internal ribosome entry site (IRES)-dependent translation. Analysis of translating ribosome composition in stressed and unstressed cardiomyocytes was conducted using mass spectrometry. We observed no significant changes in ribosomal protein composition, but several mitochondrial ribosomal proteins (MRPs) were identified in cytosolic polysomes, showing drastic variations between stressed and unstressed cells. The most notable increase in polysomes of stressed cells was observed in MRPS15. Its interaction with ribosomal proteins was confirmed by proximity ligation assay (PLA) and immunoprecipitation, suggesting its intrinsic role as a ribosomal component during stress. Knock-down or overexpression experiments of MRPS15 revealed its role as an activator of IRES-dependent translation. Furthermore, polysome profiling after immunoprecipitation with anti-MRPS15 antibody revealed that the "MRPS15 ribosome" is specialized in translating mRNAs involved in the unfolded protein response.

Spatial sequestration of activated-caspase 3 in aggresomes mediates resistance of neuroblastoma cell to bortezomib treatment.

Neuroblastoma (NB) is the most common pediatric tumor and is currently treated by several types of therapies including chemotherapies, such as bortezomib treatment. However, resistance to bortezomib is frequently observed by mechanisms that remain to be deciphered. Bortezomib treatment leads to caspase activation and aggresome formation. Using models of patients-derived NB cell lines with different levels of sensitivity to bortezomib, we show that the activated form of caspase 3 accumulates within aggresomes of NB resistant cells leading to an impairment of bortezomib-induced apoptosis and increased cell survival. Our findings unveil a new mechanism of resistance to chemotherapy based on an altered subcellular distribution of the executioner caspase 3. This mechanism could explain the resistance developed in NB patients treated with bortezomib, emphasizing the potential of drugs targeting aggresomes.

Isocitrate dehydrogenase wt and IDHmut adult-type diffuse gliomas display distinct alterations in ribosome biogenesis and 2'O-methylation of ribosomal RNA.

BACKGROUND: High-grade adult-type diffuse gliomas (HGGs) constitute a heterogeneous group of aggressive tumors that are mostly incurable. Recent advances highlighting the contribution of ribosomes to cancer development have offered new clinical perspectives. Here, we uncovered that isocitrate dehydrogenase (IDH)wt and IDHmut HGGs display distinct alterations of ribosome biology, in terms of rRNA epitranscriptomics and ribosome biogenesis, which could constitute novel hallmarks that can be exploited for the management of these pathologies. METHODS: We analyzed (1) the ribosomal RNA 2'O-ribose methylation (rRNA 2'Ome) using RiboMethSeq and in-house developed bioinformatics tools (https://github.com/RibosomeCRCL/ribomethseq-nfandrRMSAnalyzer) on 3 independent cohorts compiling 71 HGGs (IDHwt n = 30, IDHmut n = 41) and 9 non-neoplastic samples, (2) the expression of ribosome biogenesis factors using medium throughput RT-qPCR as a readout of ribosome biogenesis, and (3) the sensitivity of 5 HGG cell lines to RNA Pol I inhibitors (CX5461, BMH-21). RESULTS: Unsupervised analysis demonstrated that HGGs could be distinguished based on their rRNA 2'Ome epitranscriptomic profile, with IDHwt glioblastomas displaying the most significant alterations of rRNA 2'Ome at specific sites. In contrast, IDHmut HGGs are largely characterized by an overexpression of ribosome biogenesis factors compared to non-neoplastic tissues or IDHwt glioblastomas. Finally, IDHmut HGG-derived spheroids display higher cytotoxicity to CX5461 than IDHwt glioblastoma, while all HGG spheroids display a similar cytotoxicity to BMH-21. CONCLUSIONS: In HGGs, IDH mutational status is associated with specific alterations of the ribosome biology and with distinct sensitivities to RNA Pol I inhibitors.

Customization of the translational complex regulates mRNA-specific translation to control CNS regeneration.

In the adult mammalian central nervous system (CNS), axons fail to regenerate spontaneously after injury because of a combination of extrinsic and intrinsic factors. Despite recent advances targeting the intrinsic regenerative properties of adult neurons, the molecular mechanisms underlying axon regeneration are not fully understood. Here, we uncover a regulatory mechanism that controls the expression of key proteins involved in regeneration at the translational level. Our results show that mRNA-specific translation is critical for promoting axon regeneration. Indeed, we demonstrate that specific ribosome-interacting proteins, such as the protein Huntingtin (HTT), selectively control the translation of a specific subset of mRNAs. Moreover, modulating the expression of these translationally regulated mRNAs is crucial for promoting axon regeneration. Altogether, our findings highlight that selective translation through the customization of the translational complex is a key mechanism of axon regeneration with major implications in the development of therapeutic strategies for CNS repair.

The stress sensor GCN2 differentially controls ribosome biogenesis in colon cancer according to the nutritional context.

Nutrient availability is a key determinant of tumor cell behavior. While nutrient-rich conditions favor proliferation and tumor growth, scarcity, and particularly glutamine starvation, promotes cell dedifferentiation and chemoresistance. Here, linking ribosome biogenesis plasticity with tumor cell fate, we uncover that the amino acid sensor general control non-derepressible 2 (GCN2; also known as eIF-2-alpha kinase 4) represses the expression of the precursor of ribosomal RNA (rRNA), 47S, under metabolic stress. We show that blockade of GCN2 triggers cell death by an irremediable nucleolar stress and subsequent TP53-mediated apoptosis in patient-derived models of colon adenocarcinoma (COAD). In nutrient-rich conditions, a cell-autonomous GCN2 activity supports cell proliferation by stimulating 47S rRNA transcription, independently of the canonical integrated stress response (ISR) axis. Impairment of GCN2 activity prevents nuclear translocation of methionyl-tRNA synthetase (MetRS), resulting in nucleolar stress, mTORC1 inhibition and, ultimately, autophagy induction. Inhibition of the GCN2-MetRS axis drastically improves the cytotoxicity of RNA polymerase I (RNA pol I) inhibitors, including the first-line chemotherapy oxaliplatin, on patient-derived COAD tumoroids. Our data thus reveal that GCN2 differentially controls ribosome biogenesis according to the nutritional context. Furthermore, pharmacological co-inhibition of the two GCN2 branches and RNA pol I activity may represent a valuable strategy for elimination of proliferative and metabolically stressed COAD cells.

Correction: The interferon stimulated gene 20 protein (ISG20) is an innate defense antiviral factor that discriminates self versus non-self translation.

[This corrects the article DOI: 10.1371/journal.ppat.1008093.].

RSL24D1 sustains steady-state ribosome biogenesis and pluripotency translational programs in embryonic stem cells.

Embryonic stem cell (ESC) fate decisions are regulated by a complex circuitry that coordinates gene expression at multiple levels from chromatin to mRNA processing. Recently, ribosome biogenesis and translation have emerged as key pathways that efficiently control stem cell homeostasis, yet the underlying molecular mechanisms remain largely unknown. Here, we identified RSL24D1 as highly expressed in both mouse and human pluripotent stem cells. RSL24D1 is associated with nuclear pre-ribosomes and is required for the biogenesis of 60S subunits in mouse ESCs. Interestingly, RSL24D1 depletion significantly impairs global translation, particularly of key pluripotency factors and of components from the Polycomb Repressive Complex 2 (PRC2). While having a moderate impact on differentiation, RSL24D1 depletion significantly alters ESC self-renewal and lineage commitment choices. Altogether, these results demonstrate that RSL24D1-dependant ribosome biogenesis is both required to sustain the expression of pluripotent transcriptional programs and to silence PRC2-regulated developmental programs, which concertedly dictate ESC homeostasis.

snoDB 2.0: an enhanced interactive database, specializing in human snoRNAs.

snoDB is an interactive database of human small nucleolar RNAs (snoRNAs) that includes up-to-date information on snoRNA features, genomic location, conservation, host gene, snoRNA-RNA targets and snoRNA abundance and provides links to other resources. In the second edition of this database (snoDB 2.0), we added an entirely new section on ribosomal RNA (rRNA) chemical modifications guided by snoRNAs with easy navigation between the different rRNA versions used in the literature and experimentally measured levels of modification. We also included new layers of information, including snoRNA motifs, secondary structure prediction, snoRNA-protein interactions, copy annotations and low structure bias expression data in a wide panel of tissues and cell lines to bolster functional probing of snoRNA biology. Version 2.0 features updated identifiers, more links to external resources and duplicate entry resolution. As a result, snoDB 2.0, which is freely available at https://bioinfo-scottgroup.med.usherbrooke.ca/snoDB/, represents a one-stop shop for snoRNA features, rRNA modification targets, functional impact and potential regulators.

Low level of Fibrillarin, a ribosome biogenesis factor, is a new independent marker of poor outcome in breast cancer.

BACKGROUND: A current critical need remains in the identification of prognostic and predictive markers in early breast cancer. It appears that a distinctive trait of cancer cells is their addiction to hyperactivation of ribosome biogenesis. Thus, ribosome biogenesis might be an innovative source of biomarkers that remains to be evaluated. METHODS: Here, fibrillarin (FBL) was used as a surrogate marker of ribosome biogenesis due to its essential role in the early steps of ribosome biogenesis and its association with poor prognosis in breast cancer when overexpressed. Using 3,275 non-metastatic primary breast tumors, we analysed FBL mRNA expression levels and protein nucleolar organisation. Usage of TCGA dataset allowed transcriptomic comparison between the different FBL expression levels-related breast tumours. RESULTS: We unexpectedly discovered that in addition to breast tumours expressing high level of FBL, about 10% of the breast tumors express low level of FBL. A correlation between low FBL mRNA level and lack of FBL detection at protein level using immunohistochemistry was observed. Interestingly, multivariate analyses revealed that these low FBL tumors displayed poor outcome compared to current clinical gold standards. Transcriptomic data revealed that FBL expression is proportionally associated with distinct amount of ribosomes, low FBL level being associated with low amount of ribosomes. Moreover, the molecular programs supported by low and high FBL expressing tumors were distinct. CONCLUSION: Altogether, we identified FBL as a powerful ribosome biogenesis-related independent marker of breast cancer outcome. Surprisingly we unveil a dual association of the ribosome biogenesis FBL factor with prognosis. These data suggest that hyper- but also hypo-activation of ribosome biogenesis are molecular traits of distinct tumors.

Alteration of ribosome function upon 5-fluorouracil treatment favors cancer cell drug-tolerance.

Mechanisms of drug-tolerance remain poorly understood and have been linked to genomic but also to non-genomic processes. 5-fluorouracil (5-FU), the most widely used chemotherapy in oncology is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways. Here, we show that 5-FU treatment leads to the production of fluorinated ribosomes exhibiting altered translational activities. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts, and human tumors. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs depending on the nature of their 5'-untranslated region. As a result, we find that sustained translation of IGF-1R mRNA, which encodes one of the most potent cell survival effectors, promotes the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that "man-made" fluorinated ribosomes favor the drug-tolerant cellular phenotype by promoting translation of survival genes.

DHX30 Coordinates Cytoplasmic Translation and Mitochondrial Function Contributing to Cancer Cell Survival.

DHX30 was recently implicated in the translation control of mRNAs involved in p53-dependent apoptosis. Here, we show that DHX30 exhibits a more general function by integrating the activities of its cytoplasmic isoform and of the more abundant mitochondrial one. The depletion of both DHX30 isoforms in HCT116 cells leads to constitutive changes in polysome-associated mRNAs, enhancing the translation of mRNAs coding for cytoplasmic ribosomal proteins while reducing the translational efficiency of the nuclear-encoded mitoribosome mRNAs. Furthermore, the depletion of both DHX30 isoforms leads to higher global translation but slower proliferation and lower mitochondrial energy metabolism. Isoform-specific silencing supports a role for cytoplasmic DHX30 in modulating global translation. The impact on translation and proliferation was confirmed in U2OS and MCF7 cells. Exploiting RIP, eCLIP, and gene expression data, we identified fourteen mitoribosome transcripts we propose as direct DHX30 targets that can be used to explore the prognostic value of this mechanism in cancer. We propose that DHX30 contributes to cell homeostasis by coordinating ribosome biogenesis, global translation, and mitochondrial metabolism. Targeting DHX30 could, thus, expose a vulnerability in cancer cells.

A novel view on an old drug, 5-fluorouracil: an unexpected RNA modifier with intriguing impact on cancer cell fate.

5-Fluorouracil (5-FU) is a chemotherapeutic drug widely used to treat patients with solid tumours, such as colorectal and pancreatic cancers. Colorectal cancer (CRC) is the second leading cause of cancer-related death and half of patients experience tumour recurrence. Used for over 60 years, 5-FU was long thought to exert its cytotoxic effects by altering DNA metabolism. However, 5-FU mode of action is more complex than previously anticipated since 5-FU is an extrinsic source of RNA modifications through its ability to be incorporated into most classes of RNA. In particular, a recent report highlighted that, by its integration into the most abundant RNA, namely ribosomal RNA (rRNA), 5-FU creates fluorinated active ribosomes and induces translational reprogramming. Here, we review the historical knowledge of 5-FU mode of action and discuss progress in the field of 5-FU-induced RNA modifications. The case of rRNA, the essential component of ribosome and translational activity, and the plasticity of which was recently associated with cancer, is highlighted. We propose that translational reprogramming, induced by 5-FU integration in ribosomes, contributes to 5-FU-driven cell plasticity and ultimately to relapse.

Erratum: Ribosomal RNA 2' O-methylation as a novel layer of inter-tumour heterogeneity in breast cancer.

[This corrects the article DOI: 10.1093/narcan/zcaa036.].

Study of intracellular anabolism of 5-fluorouracil and incorporation in nucleic acids based on an LC-HRMS method.

5-Fluorouracil (5-FU) is an anticancer drug extensively used for different cancers. Intracellular metabolic activation leads to several nucleoside and nucleotide metabolites essential to exert its cytotoxic activity on multiple cellular targets such as enzymes, DNA and RNA. In this paper, we describe the development of a method based on liquid chromatography coupled with high resolution mass spectrometry suitable for the simultaneous determination of the ten anabolic metabolites (nucleoside, nucleotide and sugar nucleotide) of 5-FU. The chromatographic separation was optimized on a porous graphitic carbon column allowing the analysis of the metabolites of 5-FU as well as endogenous nucleotides. The detection was performed on an Orbitrap® tandem mass spectrometer. Linearity of the method was verified in intracellular content and in RNA extracts. The limit of detection was equal to 12 pg injected on column for nucleoside metabolites of 5-FU and 150 pg injected on column for mono- and tri-phosphate nucleotide metabolites. Matrix effect was evaluated in cellular contents, DNA and RNA extracts for nucleoside and nucleotides metabolites. The method was successfully applied to i) measure the proportion of each anabolic metabolite of 5-FU in cellular contents, ii) follow the consequence of inhibition of enzymes on the endogenous nucleotide pools, iii) study the incorporation of metabolites of 5-FU into RNA and DNA, and iv) to determine the incorporation rate of 5-FUrd into 18 S and 28 S sub-units of rRNA.

Ribosome Biogenesis Alterations in Colorectal Cancer.

Many studies have focused on understanding the regulation and functions of aberrant protein synthesis in colorectal cancer (CRC), leaving the ribosome, its main effector, relatively underappreciated in CRC. The production of functional ribosomes is initiated in the nucleolus, requires coordinated ribosomal RNA (rRNA) processing and ribosomal protein (RP) assembly, and is frequently hyperactivated to support the needs in protein synthesis essential to withstand unremitting cancer cell growth. This elevated ribosome production in cancer cells includes a strong alteration of ribosome biogenesis homeostasis that represents one of the hallmarks of cancer cells. None of the ribosome production steps escape this cancer-specific dysregulation. This review summarizes the early and late steps of ribosome biogenesis dysregulations described in CRC cell lines, intestinal organoids, CRC stem cells and mouse models, and their possible clinical implications. We highlight how this cancer-related ribosome biogenesis, both at quantitative and qualitative levels, can lead to the synthesis of ribosomes favoring the translation of mRNAs encoding hyperproliferative and survival factors. We also discuss whether cancer-related ribosome biogenesis is a mere consequence of cancer progression or is a causal factor in CRC, and how altered ribosome biogenesis pathways can represent effective targets to kill CRC cells. The association between exacerbated CRC cell growth and alteration of specific steps of ribosome biogenesis is highlighted as a key driver of tumorigenesis, providing promising perspectives for the implementation of predictive biomarkers and the development of new therapeutic drugs.

Uncovering the Translational Regulatory Activity of the Tumor Suppressor BRCA1.

BRCA1 inactivation is a hallmark of familial breast cancer, often associated with aggressive triple negative breast cancers. BRCA1 is a tumor suppressor with known functions in DNA repair, transcription regulation, cell cycle control, and apoptosis. In the present study, we demonstrate that BRCA1 is also a translational regulator. We previously showed that BRCA1 was implicated in translation regulation. Here, we asked whether translational control could be a novel function of BRCA1 that contributes to its tumor suppressive activity. A combination of RNA-binding protein immunoprecipitation, microarray analysis, and polysome profiling, was used to identify the mRNAs that were specifically deregulated under BRCA1 deficiency. Western blot analysis allowed us to confirm at the protein level the deregulated translation of a subset of mRNAs. A unique and dedicated cohort of patients with documented germ-line BRCA1 pathogenic variant statues was set up, and tissue microarrays with the biopsies of these patients were constructed and analyzed by immunohistochemistry for their content in each candidate protein. Here, we show that BRCA1 translationally regulates a subset of mRNAs with which it associates. These mRNAs code for proteins involved in major programs in cancer. Accordingly, the level of these key proteins is correlated with BRCA1 status in breast cancer cell lines and in patient breast tumors. ADAT2, one of these key proteins, is proposed as a predictive biomarker of efficacy of treatments recently recommended to patients with BRCA1 deficiency. This study proposes that translational control may represent a novel molecular mechanism with potential clinical impact through which BRCA1 is a tumor suppressor.

Ribosomal RNA 2'O-methylation as a novel layer of inter-tumour heterogeneity in breast cancer.

Recent epitranscriptomics studies unravelled that ribosomal RNA (rRNA) 2'O-methylation is an additional layer of gene expression regulation highlighting the ribosome as a novel actor of translation control. However, this major finding lies on evidences coming mainly, if not exclusively, from cellular models. Using the innovative next-generation RiboMeth-seq technology, we established the first rRNA 2'O-methylation landscape in 195 primary human breast tumours. We uncovered the existence of compulsory/stable sites, which show limited inter-patient variability in their 2'O-methylation level, which map on functionally important sites of the human ribosome structure and which are surrounded by variable sites found from the second nucleotide layers. Our data demonstrate that some positions within the rRNA molecules can tolerate absence of 2'O-methylation in tumoral and healthy tissues. We also reveal that rRNA 2'O-methylation exhibits intra- and inter-patient variability in breast tumours. Its level is indeed differentially associated with breast cancer subtype and tumour grade. Altogether, our rRNA 2'O-methylation profiling of a large-scale human sample collection provides the first compelling evidence that ribosome variability occurs in humans and suggests that rRNA 2'O-methylation might represent a relevant element of tumour biology useful in clinic. This novel variability at molecular level offers an additional layer to capture the cancer heterogeneity and associates with specific features of tumour biology thus offering a novel targetable molecular signature in cancer.

The interferon stimulated gene 20 protein (ISG20) is an innate defense antiviral factor that discriminates self versus non-self translation.

ISG20 is a broad spectrum antiviral protein thought to directly degrade viral RNA. However, this mechanism of inhibition remains controversial. Using the Vesicular Stomatitis Virus (VSV) as a model RNA virus, we show here that ISG20 interferes with viral replication by decreasing protein synthesis in the absence of RNA degradation. Importantly, we demonstrate that ISG20 exerts a translational control over a large panel of non-self RNA substrates including those originating from transfected DNA, while sparing endogenous transcripts. This activity correlates with the protein's ability to localize in cytoplasmic processing bodies. Finally, these functions are conserved in the ISG20 murine ortholog, whose genetic ablation results in mice with increased susceptibility to viral infection. Overall, our results posit ISG20 as an important defense factor able to discriminate the self/non-self origins of the RNA through translation modulation.

Emerging Role of Eukaryote Ribosomes in Translational Control.

Translation is one of the final steps that regulate gene expression. The ribosome is the effector of translation through to its role in mRNA decoding and protein synthesis. Many mechanisms have been extensively described accounting for translational regulation. However it emerged only recently that ribosomes themselves could contribute to this regulation. Indeed, though it is well-known that the translational efficiency of the cell is linked to ribosome abundance, studies recently demonstrated that the composition of the ribosome could alter translation of specific mRNAs. Evidences suggest that according to the status, environment, development, or pathological conditions, cells produce different populations of ribosomes which differ in their ribosomal protein and/or RNA composition. Those observations gave rise to the concept of "specialized ribosomes", which proposes that a unique ribosome composition determines the translational activity of this ribosome. The current review will present how technological advances have participated in the emergence of this concept, and to which extent the literature sustains this concept today.