Advanced colorectal cancer (CRC) treatments often fail due to chemoresistance and tumor recurrence, primarily driven by cancer stem cells (CSCs), which play a significant role in therapy ineffectiveness. MicroRNAs (miRNAs) play a pivotal role in regulating numerous biological functions within CSCs. Through a gain-of-function miRNA screening, we identified miRNAs that regulate the ALDEFLUOR-positive CSC population in colon cancer, with miR-4745 emerging as a promising inhibitor. Overexpression of miR-4745 reduced CSC self-renewal, diminished chemoresistance, and prevented CSC enrichment after chemotherapy in patient-derived CRC models. Mechanistically, miR-4745 directly targets the 3 UTR of kinesin-light chain 2 (KLC2) mRNA, a member of the kinesin superfamily associated with poor clinical outcomes across various cancers. siRNA-mediated KLC2 downregulation reduced ALDH-positive CSCs and suppressed sphere formation, while KLC2 overexpression promoted CSC traits. Additionally, we observed an inverse correlation between KLC2 and miR-4745 mRNA levels in different colon cancer cell culture settings. Notably, KLC2 mRNA levels increased with local CRC progression and were correlated with poor overall survival in a cohort of over 1 000 stage I to IV colorectal cancer patients. Our study uncovers a novel miR-4745-KLC2 axis that regulates CSC properties and chemoresistance in CRC, offering a promising strategy to prevent relapse and improve clinical outcomes for patients. ### Competing Interest Statement The authors have declared no competing interest. La Ligue Contre le Cancer, https://ror.org/00rkrv905 Institut National du Cancer, https://ror.org/03m8vkq32 Fondation ARC pour la Recherche sur le Cancer, https://ror.org/0489qz649 Cancéropôle Grand Sud-Ouest, https://ror.org/058h5ns38 SIRIC of Montpellier Cancer

Ribosome profiling (or ribo-sequencing) is a powerful technique that enables high-resolution analysis of active translation by sequencing ribosome-protected mRNA fragments. Developed in 2009 by Nicholas Ingolia and Jonathan Weissman, it provides direct insights into which mRNAs are being translated and at what rate, surpassing traditional transcriptomic and proteomic methods. Ribo-seq allows for precise mapping of ribosome positions, enabling detailed characterization of ribosome dynamics and the identification of alternative translation initiation sites and upstream open reading frames. The standard workflow includes key steps such as ribosome stabilization, nuclease digestion, fragment isolation, and deep sequencing. The protocol described in this chapter incorporates a polysome profiling step prior to RNase treatment, allowing, for instance, the isolation of distinct ribosome species (also known as k-somes). Ribo-seq has transformed our understanding of translational regulation and has become an essential tool for omics studies in various fields such as developmental biology, cancer biology, virology, and microbiology.

Neural crest cells contribute to craniofacial formation by differentiating into skeletogenic mesenchyme and neuro-glial lineages. Using Smart-seq2 single-cell transcriptomics, we show that mesenchymal fate commitment correlates specifically with the expression of rRNA-modifying and ribosome assembly factors, rather than structural ribosomal proteins. Notably, EMG1 and NHP2 introduce key post-transcriptional modifications into 18S rRNA, including m¹acp³ψ at U1248, which requires TSR3 for final maturation. Disrupting NHP2 or TSR3 in vitro and in vivo perturbs cranial neural crest differentiation; post-migratory temporal knockout of Polr1a or Polr1c also causes craniofacial malformations. These findings align with cell type-specific m¹acp³ψ levels during neural crest differentiation. Given the neural crest contribution to neuroblastoma, we analyze patient data to find that elevated ribosomal control and rRNA-modifying proteins predict poorer outcomes. Complementary experiments in neuroblastoma cell lines reveal functional roles for TSR3 and WDR74 in mesenchymal-like tumor states. Together, our results link rRNA modifications and ribosome assembly to fate decisions, suggesting ribosomal heterogeneity shapes both normal development and tumor progression.

RNA modification analysis by LC-MS/MS is central to epitranscriptomics, yet quantitative comparison across laboratories and instrument platforms remains poorly standardized. Here, we performed a community-driven benchmarking study during the first Human RNome Project workshop to systematically evaluate cross-platform reproducibility of ribonucleoside mass spectrometry workflows. Using the same analytical column and gradient, standardized RNA samples, and shared reagents, we compared nucleoside quantification across quadrupole, time-of-flight, and orbitrap-based LC-MS platforms employing distinct acquisition strategies. While chromatographic separation was highly reproducible across systems, nucleoside-specific MS response behavior differed substantially between platforms and limited direct comparability of relative signal intensities. These response differences varied across analytes and concentration ranges, demonstrating that harmonized chromatography alone is insufficient for transferable quantitative analysis. Stable isotope-labeled internal standard (SILIS) normalization substantially reduced platform- and method-dependent response and improved agreement for most evaluated modifications. External calibration improved agreement between qTOF and Orbitrap workflows for a subset of modifications but did not fully resolve residual intersystem differences. Based on these findings, we establish benchmark-derived recommendations for harmonized relative and absolute RNA modification quantification, including guidance for calibration design, quality control, and data reporting. Together, this work provides a methodological framework for reproducible nucleoside LC-MS/MS workflows and establishes a foundation for large-scale comparative epitranscriptomic studies.

Reproductive disorders can result from a defective action of the neuropeptide gonadotropin-releasing hormone (GnRH), the master regulator of reproduction. We have previously shown that selenoprotein T (SELENOT), a newly described thioredoxin-like selenoprotein highly expressed in endocrine and neuroendocrine cells, plays a role in hormone secretion and neuroprotection. However, whether SELENOT is involved in neuroendocrine regulation in vivo is totally unknown. We found that SELENOT deficiency in the brain impaired sexual behavior, leading to a decline in fertility in both male and female mice. Biochemical and histological analyses of the gonadotrope axis of these mice revealed a higher expression of GnRH, which is associated with circulating luteinizing hormone (LH) excess, and elevated steroid hormones in males and a polycystic ovary syndrome-like phenotype in females. In addition, SELENOT deficiency impaired LH pulse secretion in both male and female mice. These changes were reverted after administration of a GnRH antagonist. Together, our data demonstrate for the first time to our knowledge the role of a selenoprotein in the central control of sexual behavior and reproduction, and identify a redox effector of GnRH neuron activity impacting both male and female reproductive function.

The uridine at position 34 of tRNA anticodon loops is always modified at variable levels depending on environmental conditions, but a function for this highly conserved modification has not been firmly established. Using Arabidopsis thaliana , we show that the thiolation of U34 is a prerequisite for the subsequent modifications at position 32 and 37 of the tRNALys(UUU) anticodon loop, revealing a novel modification network. Surprisingly, the level of tRNALys(UUU) is strongly increased rather than reduced in the ctu1 or ctu2 mutant backgrounds that prevent these modifications. This suggests the existence of a regulatory feedback loop that drives the transcription of this specific tRNA gene family. Furthermore, we observed that the ability of the ribosome to decode AAA, GAA and CAA codons is impaired when the thiol group is lost, leading to a reduction in protein production, especially for genes enriched in these codons. Finally, we show that loss of tRNA thiolation results in variations in levels of many proteins involved in sulfur-compound metabolism and several sulfur-containing metabolites, suggesting that the level of tRNA thiolation may act as a sensor that regulates these processes. ### Competing Interest Statement The authors have declared no competing interest. Data supporting [Figure 1][1] and [2][2] (tRNA mass spectrometry) can be found in Table S1. Mim-tRNAseq datasets supporting [Figure 3][3] have been deposited in SRA, (PRJNA1330141). Data supporting the 5’P-seq results of [Figure 4][4] have been deposited in SRA, (PRJNA1327086). Data supporting the proteomic analysis of [Figure 5][5] and [6][6] have been deposited in the PRIDE database (reference: 1-20250919-080111-3723971) Agence Nationale de la Recherche, ANR-22-CE20-0023-01, ANR-18-EURE-0019, ANR-10-LABX-41 German Research Foundation (DFG), EXC 2048/1 - project 390686111 Transregional collaborative research center, TRR341/1 - project 456082119 [1]: #F1 [2]: #F2 [3]: #F3 [4]: #F4 [5]: #F5 [6]: #F6

Introduction: Large B-Cell Lymphoma (LBCL) is an aggressive and the most common non-Hodgkin lymphoma, accounting for 30% of cases. The first-line treatment consists of a R-CHOP-like regimen, where 20-40% of patients are refractory to the treatment or will relapse (R/R). Aiming to develop new therapies and identify new biomarkers, we analyzed a recently studied biological process: epitranscriptomic modifications. These chemical modifications affect RNA nucleosides, including methylation like the N6-methyladenoside (m6A), or isomerization of the uridine (Ψ, pseudouridine). These modifications are realized by epitranscriptomic enzymes called “writers” and “erasers”, and processed by “readers”. Methods: The first part of the project was exploratory, aiming to determine whether epitranscriptomic modifications play a role in LBCL, using primary tumor cells. We included 62 patients from the Hemodiag cohort (malignant hemopathies at the CHU of Montpellier), with available clinical and biological data, as well as RNA sequencing data. Mass spectrometry was performed on the RNA of the 62 samples to analyze epitranscriptomic modifications, and we correlated these findings with overall survival via MaxStat analysis. Then we worked on cell lines to validate our findings and investigate the underlying mechanisms. We used CellTiter-Glo® (CTG) and Annexin-V/7-AAD with flow cytometry for viability assays and dynamic BH3-profiling, as well as Western Blots to evaluate apoptosis and DNA damage. Results: Twelve epitranscriptomic marks were found associated with a good or a bad prognosis, including m1G, m1A (bad overall survival = OS), and i6A (good OS). We then analyzed 52 genes that code for epitranscriptomic enzymes using RNA-seq. The expression of 27 of these was significantly correlated with good OS, including TRIT1 (writer of i6A), and 4 with a bad OS, including TRMT5 (writer of m1G) and TRMT61A (writer of m1A). Based on these results, we then tested in vitro the effect of inhibiting the epitranscriptomic enzymes METTL1-WDR4, METTL3, ALKBH5, and YTHDC1 on 3 DLBCL cell lines. Using CTG, the IC50 values for YTHDC1 were 3.75, 2.63, and 2.183 µM for OCI-LY1, NUDHL1, and RI-1, respectively. Interestingly, METTL3i's IC50 on OCI-LY1 was high (32.97 µM) and non-reached (NR) on RI-1, but low on NUDHL1 (2.74 µM), showing cell specificity. IC50 were NR for METTL1i and ALKBH5i. At a dose of 3 µM and after 24 h of treatment, apoptosis was induced by the inhibition of YTHDC1, as evidenced by the cleavage of caspase 3 and PARP, confirmed by the analysis of apoptosis by Annexin-V/7-AAD. The 4 inhibitors did not induce DNA damage at a dose of 3 µM (no γH2AX and S15P53). Finally, we performed dynamic BH3-profiling to evaluate the combination effect of YTHDC1i with BH3 inhibitors on cell viability by CTG and on BH3 dependencies by flow cytometry. The results showed a synergic effect between the anti-apoptotic protein inhibitors tested and YTHDC1i, particularly with at a low dose of venetoclax (0.01 µM, BCL-2 inhibitor), with a mean of 17% of viability of OCI-LY1 cells, in comparison to venetoclax alone (mean of 35% viability). Interestingly, when the MCL-1 inhibitor (0.1 µM, AZD-5991) was combined with YTHDC1i, we reached a mean of 8% of viability in comparison to MCL-1i alone (mean of 62%). No synergic effect was found between YTHDC1i and the BCL-xL inhibitor (A-1155463). Nevertheless, no increased BH3 dependencies were found. These results will be updated if presented at the ASH meeting. Conclusion: Our study reveals that specific epitranscriptomic modifications and the expression of their regulatory enzymes are significantly associated with overall survival in patients with LBCL. Among these, i6A and its writer, TRIT1, correlate with a better prognosis, while m1G and m1A, along with their associated enzymes, predict poorer outcomes. Functional assays identified YTHDC1 as a promising therapeutic target, with its inhibition inducing apoptosis and demonstrating strong synergy with BCL-2 and MCL-1 inhibitors in DLBCL cell lines. These findings establish a novel epitranscriptomic landscape in LBCL and support the development of targeted therapies based on RNA modification profiles.

The rising incidence of cancer and the frequent resistance to treatments are driving the scientific community to explore new biological frontiers in search of concrete solutions for personalized patient care. These initiatives are made possible by the ongoing development of innovative technologies, which are shedding new light on our understanding of biological mechanisms. One such area is ribonucleic acid (RNA) chemical modifications-known as the epitranscriptome-which play a key role in all post-transcriptional stages of gene expression. An increasing number of studies are linking these modifications to tumor progression and treatment resistance. Functionally, epitranscriptomic modifications are orchestrated by a set of proteins known as "writers", "erasers" and "readers" which respectively add, remove, or read these chemical marks on RNA. The expression of these regulatory proteins is often dysregulated in cancer, thereby contributing to carcinogenesis. Clinically, these modifications are relevant across the entire patient care continuum, including diagnostic, prognostic, predictive, and therapeutic aspects. Many epitranscriptomic marks are associated with overall survival, tumor stage, the presence of metastases, or the detection of specific cancer types. They can enhance treatment efficacy or help anticipate resistance by modulating gene expression in target cells and revealing molecular signatures associated with therapeutic escape mechanisms. Moreover, inhibitors targeting epitranscriptomic regulatory proteins are currently under development and being evaluated in clinical trials, paving the way for novel therapeutic strategies in oncology.

The cornea, the transparent outermost layer of the eye, possesses exceptional wound healing capabilities essential for vision preservation. The complexity of the corneal microenvironment is central to its rapid healing; however, the molecular mechanisms orchestrating this process remain poorly defined, limiting therapeutic advancements. Here, we elucidate the extensive remodeling of the corneal molecular landscape following physical injury. Multi-omics analyses, including transcriptomic, epitranscriptomic, and proteomic profiling, uncover significant induction of epithelial cell plasticity driving wound closure. Moreover, lacrimal gland ablation further suppresses Pax6 expression, highlighting its regulatory role. Our multi-omic approach uniquely reveals bilateral remodeling of the molecular environment, a phenomenon constrained by an intact tear film. Collectively, our findings identify novel molecular factors critical to corneal healing, significantly advancing the understanding of epithelial plasticity. These insights will facilitate the translation of cell plasticity research into innovative strategies for tissue and organ regeneration. ### Competing Interest Statement The authors have declared no competing interest.

Viruses in the Mononegavirales order encode a large protein that orchestrates replication, transcription, and the capping of viral RNA. This protein, comprising over 2.000 amino acids, contains an RNA-dependent RNA polymerase, a capping domain, and a methyltransferase (MTase) domain involved in methylating the cap structure. The MTase domain features a conserved K-D-K-E catalytic tetrad -typical of 2′O-methyltransferases-which is essential for methylating viral mRNA caps at both the N7 and 2′O positions. However, the role of these residues in other epitranscriptomic modifications of rabies virus (RABV) RNAs remains poorly characterized. To further explore the role of mRNA cap methylation in the immune evasion strategies of RABV, we investigated the functional contribution of the K-D-K-E motif within the MTase domain, using the Thai isolate as a model. Using reverse genetics, we demonstrated that the mutation K1830R in the K-D-K-E tetrad of the Tha MTase domain induces changes in the methylation landscape of viral mRNAs and, intriguingly, of host mRNAs. In addition, viruses harbouring the K1830R mutation are more sensitive to interferon-α and exhibit a less pathogenic phenotype in vitro and in vivo compared to the wild-type virus. Overall, these results suggest that the regulation of viral and cellular RNA methylation landscapes plays a crucial role in controlling RABV infection. Although the exact role of these epitranscriptomic modifications is not yet fully understood, some of these methylations appear to have proviral effects and enhance viral propagation by allowing RABV to efficiently evade the host’s antiviral response. Importance This study highlights the pivotal role of the K-D-K-E catalytic domain included in the methyltransferase domain of the large protein of Rabies virus, by modelling viral RNAs with epitranscriptomic changes. For the first time, we identify specific methylations on the viral RNA, such as 2’-O and m6A methylations, which seem to enable the virus to mask its RNA and evade detection by the host’s pattern recognition receptors. These epitranscriptomic modifications affect not only viral RNAs but also cellular RNAs, underscoring a complex interplay between viral and host mechanisms. We further demonstrate that RABV harbouring an altered K-D-R-E catalytic domain, exhibit differential methylation patterns correlated with increased sensitivity to IFN and lower pathogenicity. This emphasizes the importance of this domain in virulence and immune evasion. ### Competing Interest Statement The authors have declared no competing interest. ANR- 16-CE11-0031-01 ANR-10-INBS-09

The microbiome affects eukaryotic host cells via many metabolites, including the well-studied queuine as substrate for host tRNA queuosine modification. The microbial metabolite pre-queuosine 1 (preQ1) is produced in the bacterial tRNA queuosine biosynthesis pathway, with unknown effects on host cell biology. Here we show that preQ1 strongly represses cell proliferation in both human and mouse cells. Queuine reverses this effect by competing with preQ1 to modify the same tRNA. PreQ1 is detectable in the plasma and tissues of mice, and its injection suppresses tumour growth in a mouse cancer model. Mechanistically, preQ1 reduces cognate tRNA levels specifically, as well as codon-dependent translation of housekeeping genes. We identify the endoplasmic reticulum-localized inositol-requiring enzyme 1 (IRE1) ribonuclease as the enzyme responsible for the selective degradation of preQ1-modified tRNAs on translating ribosomes. Our results identify two microbial metabolites competing for host tRNA modification, which elicits translation quality control and impacts cell proliferation.

Plasma cells are highly specialized cells representing the end stage of B cell differentiation. They play an important role in humoral immunity by synthesizing and secreting antibodies protecting the host against infections. On the transcriptional level, the differentiation of B cells into plasma cells (PC) is associated with substantial and coordinated changes in the gene expression profile, which fall into two main categories: the loss of B cell-associated transcripts and the acquisition of plasma cell gene expression program. Many recent studies have demonstrated that RNA undergoes various modifications in a manner similar to DNA. These RNA modifications play a role in many cellular and biological processes, thereby opening up an emerging research field known as epitranscriptomics. We have shown that PC generation can be modeled using multi-step culture systems where various combinations ofactivation molecules and cytokines are subsequently applied in order to reproduce the sequential cell differentiation occurring in the different organs/tissues invivo. In these culture models, memory B cells (MBCs) differentiate intoCD20low/-CD38- pre-plasmablasts (prePBs), CD20-CD38+CD138- plasmablasts (PBs), CD20-CD38+CD138+ early PCsand, finally, into long-lived PCs (LLPCs), which may survive and produce continuously high amounts of immunoglobulins(Igs) for months invitro. Using this model coupled with single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq), we have provided significant insights into the trajectories of PC differentiation. Our findings, published by Alaterre et al. in Blood (2024), elucidated the epigenetic and transcriptional reprogramming events underpinning PC differentiation under physiological conditions. Thanks to these data we also found significant expression changes of enzymes involved in RNA editing especially enzymes implicated in N6-methyladenosine (m6A) management during the PC differentiation. This is the most common RNA modification that can affect different molecular and cellular process. A few recent studies describe the importance of m6A RNA modification for Early B cell differentiation but investigations during the latest stage of human PC differentiation remain poor. We performed RNA Mass-spectrometry analysis from each cell populations and identified that the total level of m6A mark increase during B to PC differentiation. m6A-Methylated RNA immunoprecipitation sequencing (MeRIPseq) allowed the analysis of all the transcripts that are modified by m6A mark, as well as m6A position together with the percentage of transcript modified for a given transcript. We found more than 75 000 m6A-mRNA-associated modifications and 20 000 for LncRNA with a majority specifically enriched in the preplasmablastic stage. Among m6A-mRNA that are differentially modified during B to PC differentiation we found transcript directly involved in PC differentiation and biological functions such as JCHAIN, IRF4, PRDM1, BTF3, IRF4, AICDA, but also some transcripts involved in metabolism (CD38, ARG2, FASTKD1, AK4), and DNA recombination and repair highlighting the importance of m6A mark management during B to PC differentiation. In order to investigate the role of m6A modification during PC differentiation, we used a specific inhibitor of the m6A methyltransferase METTL3, that is the main m6A-RNA transferase. We found that the inhibition of m6A deposit leads to a significant impediment of PC differentiation. METTL3 inhibitor affects plasma cell differentiation through inhibition of maturation after the preplasmablastic stage. At the cellular level, the percentage of preplasmablasts at day 7 is significantly higher compared to the control suggesting an inhibition of the maturation in plasmablasts and PCs. These results correlates with the fact that the preplasmablasts express the highest level of the enzymes involved in m6A management, such as METTL3 but also the demethylase FTO (Fat mass and Obesity-associated) and several specific readers like YTHDC1, underlining the importance of m6A-RNA modifications in the preplasmablastic stage. Single-cell RNAseq analysis of METTL3i-treated cells coupled with proteomic analysis are currently ongoing to decipher the molecular and transcriptional circuits associated with this maturation block mediated by m6A inhibition. Overall this comprehensive analysis will enhance our understanding of the role of m6A during B to PC differentiation.

Non-small cell lung cancers (NSCLCs) treated with tyrosine kinase inhibitors (TKIs) of the epidermal growth factor receptor (EGFR) almost invariably relapse in the long term, due to the emergence of subpopulations of resistant cells. Through a DNA barcoding approach, we show that the clinically approved drug sorafenib specifically abolishes the selective advantage of EGFR-TKI-resistant cells, while preserving the response of EGFR-TKI-sensitive cells. Sorafenib is active against multiple mechanisms of resistance/tolerance to EGFR-TKIs and its effects depend on early inhibition of MAPK-interacting kinase (MKNK) activity and signal transducer and activator of transcription 3 (STAT3) phosphorylation, and later down-regulation of MCL1 and EGFR. Using different xenograft and allograft models, we show that the sorafenib-EGFR-TKI combination can delay tumor growth and promote the recruitment of inflammatory cells. Together, our findings indicate that sorafenib can prolong the response to EGFR-TKIs by targeting NSCLC capacity to adapt to treatment through the emergence of resistant cells.

Mass spectrometry has become indispensable in studying post-transcriptional modifications of RNA, a.k.a. epitranscriptomics, which are crucial for regulating RNA metabolism, gene expression, and major biological processes. Over 150 chemical modifications have been identified across all RNA subtypes. Dysregulation of RNA mark deposition can lead to disease development and progression. Therefore, accurately detecting and characterizing these modifications are vital for understanding their functional impact and uncovering potential therapeutic and diagnostic biomarkers. In this chapter, we describe a mass spectrometry-based method for detecting and quantifying nearly 40 different RNA modifications.

L’incidence croissante des cancers et leur résistance aux traitements incitent la communauté scientifique à explorer de nouveaux territoires biologiques, en quête de perspectives concrètes pour la prise en charge personnalisée des patients. Ces initiatives sont rendues possibles par le développement constant de technologies innovantes, apportant une lumière nouvelle sur notre compréhension des mécanismes du vivant. C’est le cas des modifications chimiques de l’acide ribonucléique (ARN), ou épitranscriptome, qui jouent un rôle clé dans l’ensemble des étapes post-transcriptionnelles de l’expression génique. Un nombre d’études croissant associe ces modifications avec la progression tumorale et la résistance au traitement. D’un point de vue fonctionnel, les modifications épitranscriptomiques sont orchestrées par un ensemble de protéines appelées « writers », « erasers » et « readers », qui respectivement ajoutent, retirent ou interprètent ces marques chimiques sur l’ARN. L’expression de ces régulateurs est fréquemment altérée dans les cancers, contribuant ainsi à la carcinogenèse. Sur le plan clinique, ces modifications jouent un rôle à chaque étape du parcours du patient, tant au niveau diagnostique que pronostique, prédictif et thérapeutique. De nombreuses marques épitranscriptomiques sont, en effet, corrélées à la survie globale, au stade tumoral, à la présence de métastases ou encore à l’identification de certains types de cancer. Elles peuvent renforcer l’efficacité des traitements ou en anticiper la résistance, en modulant l’expression génique des cellules tumorales et en révélant des signatures moléculaires associées aux mécanismes d’échappement thérapeutique. Par ailleurs, des inhibiteurs ciblant les protéines régulatrices de l’épitranscriptome sont actuellement en développement et font l’objet d’essais cliniques, ouvrant la voie à de nouvelles approches thérapeutiques en oncologie.

Pyruvate metabolism defects lead to severe neuropathies such as the Leigh syndrome (LS) but the molecular mechanisms underlying neuronal cell death remain poorly understood. Here, we unravel a connection between pyruvate metabolism and the regulation of the epitranscriptome that plays an essential role during brain development. Using genetically engineered mouse model and primary neuronal cells, we identify the transcription factor E4F1 as a key coordinator of AcetylCoenzyme A (AcCoA) production by the pyruvate dehydrogenase complex (PDC) and its utilization as an essential co-factor by the Elongator complex to acetylate tRNAs at the wobble position uridine 34 (U34). E4F1-mediated direct transcriptional regulation of Dlat and Elp3, two genes encoding key subunits of the PDC and of the Elongator complex, respectively, ensures proper translation fidelity and cell survival in the central nervous system (CNS) during mouse embryonic development. Furthermore, analysis of PDH-deficient cells highlight a crosstalk linking the PDC to ELP3 expression that is perturbed in LS patients.

Rapid and efficient epithelial regeneration is fundamental for tissue homeostasis and proper function. As the outermost ocular structure, the cornea is transparent, multilayered, and vital for clear vision. Due to its exposed position, the cornea frequently undergoes various forms of injury affecting either the epithelium itself or its surrounding microenvironment, including corneal innervation and the tear film. Corneal abrasion, occurring commonly through trauma or as part of refractive surgical procedures, is typically viewed as a minor event since it usually resolves rapidly. Consequently, the cornea serves as an excellent model for studying epithelial wound healing. However, complications such as persistent epithelial defects or corneal opacity can develop, underscoring critical gaps in understanding the underlying molecular mechanisms. Utilizing a unilateral corneal abrasion mouse model, we conducted a comprehensive multi-omics analysis, integrating transcriptomics, proteomics, and epitranscriptomics, to dissect the dynamic molecular responses post-injury in both wounded and contralateral tissues. To elucidate the role of the tear film, we performed additional studies involving lacrimal gland ablation combined with corneal injury. We applied RNA sequencing to profile transcriptomic changes in corneal and lacrimal gland tissues, and mass spectrometry to study tear proteomics and epitranscriptomic modifications. We revealed a major modulation of the cornea transcriptome after abrasion, suggesting a regulation of pathways including JAK-STAT, Wnt and TGF-β, and a reduction of nucleoside modifications. The lacrimal gland transcriptome and tears proteome were also significantly affected. Plus, we highlighted a bilateralization, both in the cornea transcriptome and tears proteome. In the tear-deficient conditions, the wound closure rate and molecular responses were altered, and the bilateralization was impacted, with an increased matrix remodeling and a modulation of keratins expression. Our multi-omics analyses revealed extensive epithelial cellular plasticity as a key mechanism driving rapid wound closure, characterized by profound remodeling of transcriptional networks and RNA modifications. Importantly, we uncovered a previously underappreciated role of the lacrimal gland and tear film in mediating bilateral molecular responses following unilateral injury, emphasizing their pivotal roles in tissue regeneration. Additionally, we identified novel regulatory roles for RNA methylation events and critical signaling pathways implicated in epithelial healing.

During corticogenesis, projection neurons migrate along the radial glial axis to form cortical layers, the alteration of which is associated with functional deficits in adulthood. As byproducts of cell metabolism, reactive oxygen species act as second messengers to contribute to neurodevelopment; however, free radical excess may impede this process. Selenoprotein T (SELENOT) is a newly identified thioredoxin-like enzyme of the endoplasmic reticulum abundantly expressed during embryogenesis whose gene disruption in the brain leads to neuroblast cell demise and neuromorphological alterations due to increased free radical levels. To determine the potential contribution of SELENOT to the establishment of cortical networks, we first analyzed its expression profile in the neocortex at different stages of development using RNA scope in situ hybridization. These studies revealed the expression of SELENOT in different cortical layers, and its localization in glutamatergic and GABAergic neurons. Targeted SELENOT gene knockout in the cortex using in utero electroporation-mediated gene disruption or Nes-Cre/loxP transgenesis system resulted in an alteration of neuroblast migration polarity, at the level of radial scaffolding, and projection neuron positioning. These results indicate that SELENOT which is highly expressed in the cortex during neurodevelopment plays a crucial role in corticogenesis by promoting projection neuron migration.

Tumor initiating cells (TICs) are the roots of current shortcomings in advanced and metastatic cancer treatment. Endowed with self-renewal and multi-lineage differentiation capacity, TICs can disseminate and seed metastasis in distant organ. Our work identified streptomycin (SM), a potent bactericidal antibiotic, as a molecule capable of specifically targeting non-adherent TIC from colon and breast cancer cell lines. SM induces iron-dependent, reactive oxygen species (ROS)-mediated cell death, which is mechanistically distinct from RSL3-induced ferroptosis. SM-induced cell death is associated with profound alterations in mitochondrial morphology. This effect results from COX1 inhibition, which disrupts the regulation of the cytochrome c oxidase complex and triggers mitochondrial ROS production. SM's aldehyde group is essential, as its reduction into dihydrostreptomycin (DSM) abolishes its activity. These findings reveal a mechanism of action for streptomycin, shedding light on TIC metabolism and resistance, with potential implications for advanced cancer treatment.

Donor–recipient sex mismatch is an under-recognized risk factor for corneal graft rejection, yet the biological underpinnings at the ocular surface remain undefined. We hypothesized that sex hormones coordinately tune the tear–nerve–epithelium axis and that androgen exposure could normalize sex-linked differences relevant to alloimmune risk. We profiled 12-week-old mice in three hormonal contexts, i.e. male, female, and testosterone-treated female, combining tear biochemistry and label-free proteomics with epithelial lineage dynamics, in vivo sensory function, and corneal/trigeminal transcriptional readouts. Tear collection rate was comparable across groups, but total tear protein was reduced in females. Proteomics revealed extensive sex differences in extracellular composition, spanning lipid transport, protease–antiprotease balance, complement activity, and secretory/mucin pathways. Epithelial analyses showed sex-linked differences in progenitor output and spatial deployment, and sensory metrics indicated divergent innervation architecture and function. Across modalities, androgen supplementation in females shifted molecular and physiological profiles toward the male state, attenuating nearly all dimorphic signals, demonstrating that close to all sexual dimorphism observed here is reversible by testosterone and reflects an actively maintained endocrine state rather than a fixed developmental program. Complementary metabolite profiling provides a sex-stratified atlas of free modified nucleosides in tears, positioning extracellular epitranscriptomic markers as accessible indicators of hormonal context. Together, these results establish a multiscale, hormone-responsive sexual dimorphism at the ocular surface and offer a mechanistic framework linking sex and endocrine status to parameters that influence graft integration. They motivate sex-aware biomarkers, stratification by hormonal context in diagnostics and trials, and therapeutic strategies, including androgenic modulation, to mitigate the elevated rejection risk associated with sex mismatch in corneal transplantation. ### Competing Interest Statement The authors have declared no competing interest. ATIP-Avenir, 2018