Among the regulatory mechanisms that control gene expression, mRNA translation plays a fundamental role in determining the functional output of the transcriptome in cells. This is particularly relevant in immune cells, where rapid and tightly regulated translation is essential for maintaining immune homeostasis. Analyzing the translational status of transcripts, as well as the mechanisms underlying their regulation, can provide valuable insights into the key factors influencing physiological and pathological phenotypes. Here, we describe our routinely used protocol for polysome profiling, a method that enables the analysis of mRNA translation at both the individual transcript and genome-wide levels.

Uncovering novel mechanisms controlling DNA damage response (DDR) is essential to better understand the molecular features of high-grade serous ovarian cancers (HGSOC), which remain a clinical challenge. Here, we demonstrate that the glutaminyl-tRNA-synthetase QARS is a key player in DDR. We show that QARS is regulated at translational level by the MAPK pathway in HGSOC and controls homologous recombination (HR) through a translation-independent mechanism. Analyses combining RNA sequencing, immunohistochemistry, proteomic data and functional assays from HGSOC patient cohorts and relevant cellular models reveal that QARS promotes DNA repair via the HR pathway. Interestingly, we uncover that QARS is located in the nucleus and is enriched at chromatin upon DNA damage. Nuclear QARS controls HR by interacting with the nuclear autoantigen SP100, a major component of PML-nuclear bodies (PML-NB), that are regulatory hubs for DNA repair proteins. Our data highlight nuclear functions and mechanisms for QARS involving SP100, PML-NB and MRE11 that enhance HR in HGSOC. Here, the authors find that the glutaminyl-tRNA synthetase QARS, a cytosolic protein involved in mRNA translation, relocates to the nucleus to promote homologous recombination (HR). QARS promotes HR by interacting with SP100 to maintain PML-NB integrity and MRN complex localization, thus reducing tumor sensitivity to treatment.

Abstract During their inevitable evolution towards acquired resistance to anti-cancer targeted therapies, cancer cells adopt distinct gene expression profiles that allow them to transiently adapt to and tolerate the treatment. Cancer cells surviving therapy can increase their mutation rate, enhancing the likelihood of acquiring resistance-conferring mutations and evolving into resistant cells. Here we show that translational control mediates the adaptive mutability of melanoma drug-tolerant cells by regulating the translation of the error-prone non-homologous end joining (NHEJ) component 53BP1 . The specific inhibition of 5’UTR-driven 53BP1 mRNA translation was sufficient to impair NHEJ and mutability. We found that the eIF4A RNA helicase, regulates 53BP1 mRNA translation. Consequently, targeting the eIF4A with two small molecule inhibitors significantly delays the acquisition of resistance to combination of BRAF and MEK inhibitors in BRAF V600 -mutant melanoma xenograft models and cell lines by reducing the mutability of drug-tolerant cells. Our results demonstrate that a standard-of-care therapy for melanoma, by engaging non-genetic adaptation driven at the translational level, contributes to the evolution of drug-tolerant melanoma cells toward acquired resistance.

The role of mRNA translation and decay in the genotoxic stress response remains poorly explored. Here, we identify the role of yeast RGG motif-containing RNA binding protein Scd6 and its human ortholog LSM14A in genotoxic stress response. Scd6 localizes to cytoplasmic puncta upon cell treatment with various genotoxic agents. Scd6 genetically interacts with SRS2, a DNA helicase with an anti-recombination role in DNA damage repair under HU stress. Scd6 directly interacts with the SRS2 mRNA to repress its translation in cytoplasmic granules upon HU stress in an eIF4G1-independent manner. Scd6-SRS2 interaction is modulated by arginine methylation and the LSm-domain of Scd6, which acts as a cis-regulator of Scd6 arginine methylation. LSM14A regulates the translation of mRNAs encoding key NHEJ (Non-homologous end-joining) proteins such as RTEL1 (SRS2 functional homolog) and LIG4. NHEJ activity in yeast and mammalian cells is regulated by Scd6 and LSM14A, respectively. Overall, this report unveils the role of RNA binding proteins in regulating the translation of specific mRNAs coding for DNA damage response proteins upon genotoxic stress.

Although metabolic benefits of glycolysis have been extensively described in tumor cells, the extra-metabolic functions linked to this energetic pathway in tumor growth and cell proliferation have not been clearly established yet. Recently, some key glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase 2, were reported to regulate mRNA translation. Translational control of gene expression is considered as a critical effector in cancer biology, representing a highly promising area of research. Here, we report that Hexokinase 2 (HK2), a glucose kinase that catalyzes the first step of glycolysis at the outer mitochondrial membrane (OMM), is an RNA-binding protein (RBP) that regulates mRNA translation in melanoma cell lines. Polysome profiling experiments followed by RNA sequencing indicate that the translational regulation exerted by HK2 is partly independent of the metabolic status or the glycolytic pathway. We found that HK2 specifically regulates translation of the mRNA encoding SOX10, a transcription factor implicated in the regulation of tumor initiation, maintenance, and progression in melanoma. RNA-protein interaction assays, including CrossLinking ImmunoPrecipitation (CLIP), indicate that HK2 is an RBP whose interaction with RNA is independent of its enzymatic activity, its ability to bind glucose or its association with the OMM. HK2 directly interacts with the 5' untranslated region (5'UTR) of the SOX10 mRNA through a stem-loop RNA secondary structure. Using RNA-protein proximity ligation assays and a fluorescence-based ribosome-bound mRNA mapping method, we found that high glucose conditions, which promote the release of HK2 from the OMM, induce an increase in HK2-SOX10 mRNA interaction and SOX10 mRNA translation in the cytoplasm. We further showed that HK2-dependent SOX10 mRNA translation is involved in melanoma cell proliferation and colony formation. Collectively, our data highlight a nonmetabolic function of HK2 acting as an RBP and translation regulator.

The eukaryotic initiation factor 4F (eIF4F) complex is essential for the selective translational control of protein expression enabling cells to rapidly adapt to diverse stimuli. We previously identified an eIF4F/STAT1/PDL1 axis in melanoma cells. The effectiveness of cancer immunotherapy, particularly immune checkpoint inhibitors (ICIs) or tumor-infiltrating lymphocytes (TILs)-based adoptive therapy, is hindered by resistance mechanisms associated with the terminal exhaustion of T cells. Here, we investigate the role of eIF4F in the translational control of immune checkpoints (ICs) and associated transcription factors in both circulating T cells and TILs from melanoma patients, aiming to reveal potential connections between eIF4F-mediated translational regulation and T cell exhaustion. Polysome profiling was conducted on both circulating lymphocytes and TILs isolated from 10 melanoma specimens. We examined the expression of various activation/exhaustion markers by FACS, assessed T cell functions and determined cytokine secretion profiles. These experiments were performed with and without inhibition of the eIF4F-dependent translation using three different inhibitors of the eIF4A helicase (eIF4A-i: silvestrol, EFT226 and RBX0901). The effects of eIF4A-i combined with PD1 blockade were assessed in ex-vivo co-cultures, organotypic models and three syngeneic murine cancer models of melanoma and colon cancer. Key negative immune checkpoints and transcription factors including TOX, NFAc2, CTLA-4, LAG3, and TIM3 are regulated by eIF4F in TILs and can be efficiently downregulated by eIF4A-i. Notably, the factors controlled by eIF4F in circulating CD8+ T cells differ from those in TILs emphasizing the importance of patients’ tumor-derived samples. TILs proliferation is increased by eIF4A-I in vitro. Co-culture experiments with TILs and syngeneic melanoma cells along with organotypic melanoma models, showed enhanced melanoma killing by TILs when anti-PD1 was combined with eIF4A-i. Additionally a synergistic effect of eIF4A and anti-PD1 was observed in the three immunocompetent murine models tested. Our findings unveil a previously unrecognized role of eIF4F in orchestrating the translational regulation of key proteins driving T cell exhaustion. This study is the first to demonstrate that targeting eIF4F with eIF4A inhibitors not only downregulates exhaustion markers but also synergistically enhances the efficacy of anti-PD-1 therapy across multiple models, including human TILs and murine cancer systems. This novel insight underscores the immense therapeutic potential of combining eIF4A inhibitors with ICIs to overcome resistance mechanisms and reinvigorate anti-tumor immunity. Samad Muhammadnejad, Monireh Kazemimanesh, Naima Benannoune, Sandrine Agoussi, Cecile Badoual, Louis Vaquier, Helene Lecourt, stéphan Vagner, caroline Robert. Targeting eIF4F-mediated translation to reduce T cell exhaustion and enhance PD-1 blockade efficacy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4444.

BACKGROUND:Many transcript isoforms generated by intronic polyadenylation (IPA) encode isoforms of canonical proteins. Microproteins are an emerging class of small proteins translated from small open reading frames (sORFs) in noncoding RNAs and mRNAs, but their production by IPA isoforms is unknown. RESULTS:Here, by crossing 3'-seq, Ribo-Seq, and mass-spectrometry data, we identify 297 genes with a microprotein-coding IPA isoform terminating in a 5'UTR intron (coined miP-5'UTR-IPA isoform). By 3'-seq and long-read RNA-seq analyses in lung cancer cells treated with cisplatin, a DNA-cross-linking anticancer drug, we find that cisplatin globally favors the expression of (miP-5'UTR-)IPA isoforms relative to full-length mRNAs, mainly by decreasing the latter through an inhibition of transcription processivity in a FANCD2 and senataxin-dependent manner. The cisplatin-regulated miP-5'UTR-IPA isoform in the PRKAR1B gene is translated, as it is associated with light polysome fractions and contains Ribo-Seq-supported sORFs in its alternative last exon, and the microprotein (PRKAR1B-IPA-miP2) encoded by its sORF#2 is detected by Western blot and immunofluorescence. CRISPR editing of either the IPA site or the sORF#2 initiation site leads to decreased cell growth inhibition by cisplatin and camptothecin, another genotoxic drug. Mechanistically, PRKAR1B-IPA-miP2 promotes p53 protein induction by cisplatin. Finally, 70 miP-5'UTR-IPA isoforms are detected in normal cells, and 143 are upregulated by cisplatin. CONCLUSIONS:Here, we show that IPA isoforms are a novel source of microproteins, and we reveal the novel paradigm of miP-5'UTR-IPA genes that produce both a canonical full-length mRNA and a microprotein-coding IPA isoform.

Metabolism involves a wide range of pathways and chemical reactions catalysed by specialized enzymes whose activity is fundamental for living cells. In the past three decades, metabolic enzymes have emerged as critical regulators of gene expression, thus revealing unexpected functions beyond their canonical metabolic roles. In this Review, we discuss the evidences that these enzymes, with a particular focus on enzymes participating in the glucose metabolism, can directly bind RNA. This binding has been recurrently shown to be involved in the post-trasncriptional gene regulation, by influencing processes such as RNA stability, localization, translation, and degradation. Although the mechanisms underlying RNA-enzyme interactions and their regulation are still not fully elucidated, several reports suggest that some of these interactions can be influenced by substrates, metabolites, and cellular metabolic states. In contrast, direct and specific binding of RNAs was also shown to regulate the activity, stability, interaction and localization of the enzymes. The discovery of the non-canonical RNA-binding activity of metabolic enzymes not only expands our understanding of these seemingly well-characterized proteins, but also provides new perspectives on the integration of metabolic and gene regulatory networks, besides revealing potential therapeutic vulnerabilities.

Abstract Multiprotein complexes are key molecular switches of oncogenic signal transduction pathways that relay the flux of information coming from transmembrane receptors and distribute signals to a myriad of effectors. As such the regulation of protein-protein interactions is crucial in signal transduction pathways. For instance, activation of the mitogen-activated protein kinases pathway (MAPK) occurs through a cascade of phosphorylation and protein-protein interactions (PPIs) and is often deregulated in cancer. RNA-protein interactions play key roles in biological processes. More than a thousand RNA binding proteins (RBPs) influence the fate of mRNAs and non-coding RNAs in different ways. It is however less described how RNAs can in turn influence the functions of proteins to which they bind. Here we propose that RNAs scaffold PPIs in the MAPK pathway, thereby fine-tuning oncogenic signaling. We focused our study on cutaneous melanoma for which the MAPK is activated in more than 70% of the cases due to activating mutations in BRAF and NRAS. Using UV-CrossLinking ImmunoPrecipitation (CLIP) experiments, we found that several proteins of the MAPK pathway, including BRAF (but not MEK and ERK) interact with RNAs in RAS-activated melanoma cells. In addition, using a PLA- based imaging technique that allows the visualisation of the proximity of two given proteins, we showed that RNAs promote the stabilisation of BRAF-PPIs, suggesting a scaffolding role of RNAs in the MAPK pathway. Loss of BRAF RNA binding activity (achieved with a single point mutation) is associated with decreased BRAF dimerization and signaling. The oncogenic mutant protein BRAF (V600E) also interacts with RNA in a variety of melanoma cell lines including a BRAF inhibitor-resistant melanoma cell line in which the dimerization of BRAF and CRAF is partly dependent on RNA. Identifying BRAF-bound RNAs will open new therapeutic strategies targeting RNA-protein interactions. We also envision that the level of BRAF-RNA interactions could serve as a predictive marker of response to BRAF (V600E) inhibitors. Citation Format: Alexia Le Barch, Sabrina Mennour, Patricia Uguen, Stephan Vagner. The RNA binding activity of the BRAF Kinase [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B009.

Several studies have reported the importance of aerobic glycolysis in melanoma development. Although metabolic benefits of glycolysis have been extensively described in tumor cells, the extra-metabolic functions linked to this energetic pathway in melanoma growth and proliferation have not been clearly established yet. Recently, some key glycolytic enzymes, such as GAPDH and PKM2, were reported to regulate mRNA translation. Translational control of gene expression is considered as a critical effector in cancer biology, representing a highly promising area of research. Here, we report that Hexokinase 2 (HK2), a glucose kinase that catalyzes the first step of glycolysis, is an RNA binding protein (RBP) that regulates mRNA translation in melanoma. We show that siRNA-mediated HK2 depletion changes the translational landscape of melanoma cells. Polysome profiling experiments and RNA-Seq indicate that the translational regulation exerted by HK2 is partly independent of the metabolic status or the glycolytic pathway. We found that HK2 specifically regulates the translation of the mRNA encoding SOX10, a transcription factor implicated in the regulation of tumor initiation, maintenance and progression in melanoma. RNA-protein interaction assays, including crosslinking immunoprecipitation (CLIP), indicate that HK2 is an RBP whose interaction with RNA is independent of its hexokinase activity or subcellular localization. We also show that HK2 specifically associates with the 5 prime untranslated region (5 prime UTR) of the SOX10 mRNA, and that several deletions in this region decreases both HK2-SOX10 mRNA association and SOX10 5 prime UTR-mediated translation. We further show that HK2-dependent SOX10 translational regulation is involved in melanoma cell proliferation and colony formation. Collectively, our data highlight a non-metabolic function of HK2, indicating that melanoma cells may enhance glycolysis for purposes beyond simple anabolism. ### Competing Interest Statement The authors have declared no competing interest.

Amino acid bioavailability impacts mRNA translation in a codon-dependent manner. Here, we report that the anti-cancer MAPK inhibitors (MAPKi) decrease the intracellular concentration of aspartate and glutamate in melanoma cells. This coincides with the accumulation of ribosomes on codons corresponding to these amino acids and triggers the translation-dependent degradation of mRNAs encoding aspartate- and glutamate-rich proteins, involved in DNA metabolism such as DNA replication and repair. Consequently, cells that survive MAPKi degrade aspartate and glutamate likely to generate energy, which simultaneously decreases their requirement for amino acids due to the downregulation of aspartate- and glutamate-rich proteins involved in cell proliferation. Concomitantly, the downregulation of aspartate- and glutamate-rich proteins involved in DNA repair increases DNA damage loads. Thus, DNA repair defects, and therefore mutations, are at least in part a secondary effect of the metabolic adaptation of cells exposed to MAPKi.

A better understanding of the RNA biology and chemistry is necessary to then develop new RNA therapeutic strategies. This review is the synthesis of a series of conferences that took place during the 6th international course on post-transcriptional gene regulation at Institut Curie. This year, the course made a special focus on RNA chemistry.

Genotoxic stress, arising from various environmental sources and endogenous cellular processes, pose a constant threat to genomic stability. Cells have evolved intricate mechanisms to detect and repair DNA damage, orchestrating a robust genotoxic stress response to safeguard the integrity of the genome. Recent research has shed light on the crucial role of co- and post-transcriptional regulatory mechanisms in modulating the cellular response to genotoxic stress. Here we highlight recent advances illustrating the intricate interplay between pre-mRNA processing, with a focus on 3'-end processing, and genotoxic stress response.

RNA-binding proteins (RBPs) are involved in many biological processes. The direct interaction between protein and RNA can be studied using cross-linking immunoprecipitation (CLIP) techniques in living cells. Here, we present a protocol to characterize the direct binding of proteins to RNA:DNA hybrids or RNA-DNA chimeras in living cells using CLIP. We describe steps for RNA-protein UV-C cross-linking in living cells, isolating RNA-protein complexes, RNA labeling, and extracting nucleic acid. We then detail procedures for nuclease treatment and nucleic acid migration.

OBJECTIVES:The RNA epitranscriptomic modification known as N6-methyladenosine (m6A) represents a novel mechanism of gene regulation that is poorly understood in human autoimmune diseases. Our research explores the role of this RNA m6A modification in salivary gland epithelial cells (SGEC) and its impact on the pathogenesis of Sjögren's disease (SjD). METHODS:SGECs from SjD patients and controls were analysed for m6A writers METTL3 and METTL14 expression using RNA-seq, quantitative PCR and immunohistochemistry. Functional assays assessed the impact of METTL3 knockdown or pharmacological inhibition on proinflammatory gene expression and immune cell interactions (using transwell and coculture systems). Mechanistic studies examined METTL3-mediated m6A modifications in double-stranded RNA (dsRNA) formation through immunofluorescence. Unsupervised clustering identified patterns of interferon activation in salivary glands and their correlation with m6A writers. RESULTS:METTL3 and METTL14 were elevated in SGEC from SjD patients in comparison to controls. Paradoxically, inhibiting METTL3 increased proinflammatory gene expression, enhancing SGEC's ability to attract immune cells and activate B cells. Conversely, inhibiting the eraser FTO had the opposite effect. METTL3-mediated m6A modifications prevented dsRNA formation and IFN signalling activation. SGEC from SjD showed insufficient METTL3 upregulation compared with controls in response to inflammatory triggers, indicating a limited capacity to regulate the inflammatory response. SjD patients with elevated disease activity and higher interferon signature exhibit reduced METTL3 expression. CONCLUSIONS:Impairment of m6A modifications in SGEC in response to inflammatory triggers favour the formation of dsRNA, potentially amplifying the interferon loop and contributing to SjD pathogenesis.

Supplementary Table 3 from Widespread Estrogen-Dependent Repression of microRNAs Involved in Breast Tumor Cell Growth