Epigenetic RNA modifications play important roles in the regulation of gene expression. Canonical cap-dependent translation relies on the presence of a 7-methylguanosine (m7G cap) at the 5’ end of mRNAs to recruit the initiation factor eIF4E. The strict dependence on the eIF4E/m7G interaction is called into question by multiple transcriptomic analyses, challenging the classical cap-dependent translation model. We reported the first example of epigenetic modification of the cap of certain mRNAs in mammals and showed that several stress-related selenoprotein mRNAs have a hypermodified m32,2,7G cap (or TMG cap). These mRNAs are not recognized by eIF4E but are nevertheless translated by a mechanism that needs to be elucidated. We recently identified the entire repertoire of TMG-capped mRNAs at the transcriptome level by TMG immunoprecipitation coupled to RNA-seq (TMG RIP-seq). TMG-capped mRNA genes encode proteins involved in translation, stress response, antioxidant protection, and cancer prevention. Our hypothesis is that the TMG cap may allow mRNAs to be directed to specific translation pathways. The aim of the ReCAPtrans project is to determine the role of the TMG cap in mRNA translation and localization, identify associated translation factors, and characterize the repertoire of TMG-capped mRNAs under stress conditions to understand the role of this modification in the regulation of translation. The role of the TMG cap in endogenous TMG-capped mRNA translation will be studied by the combination of TMG IP and polysome fractionation in canonical conditions and torin-1 inhibition that inactivates eIF4E-dependent translation. Translation of selected synthetic TMG- or m7G-capped mRNA transcripts will be used to decipher translation mechanisms in vitro using cell-free translation assays, or in vivo. TMG caps identified in noncoding RNAs play a role in their functional localization. The impact of the TMG cap on mRNA localization will be analyzed by subcellular fractionation and immunolocalization. The potential role of TMG-cap binding proteins, known for their role in nucleocytoplasmic trafficking, will be evaluated. To capture native initiation factors involved in TMG-capped mRNA translation, we will use a ribosome/mRNA particle purification method dedicated to in-depth proteomic analysis. This will provide access to the composition of the translation complexes used by these different mRNAs, cap-binding factors and auxiliary proteins. Validation of trans-acting factors will be performed by classical mRNA-protein or protein-protein interaction assays. Their impact on the translation of TMG-capped mRNAs will be tested by siRNA. Particular attention will be paid to RNA binding proteins previously identified to support alternative cap-dependent translation such as initiation factors eIF4G2, eIF3 and cap binding proteins. TMG-RIP seq experiments will be performed in different cell types (HEK293FT, HeLa or HEPG2) and under various conditions of stress (oxidative stress, hypoxic conditions, amino acid starvation) to determine whether the repertoire of TMG-capped mRNAs is regulated and could thus modulate translation under certain conditions. This will indicate if a common model for their translation emerges or if specific mRNA pathways are engaged. Epigenetic modifications of the mRNA cap have never been studied. ReCAPtrans should elucidate a new mechanism of translation regulation in eukaryotes. Translational responses triggered by cellular stress will be particularly relevant for diseases such as cancer. Wether translational responses are triggered by cellular stress will be particularly relevant for diseases such as cancer. The ReCAPtrans proposal should represent a step forward in the characterization of new post-transcriptional control mechanisms in higher eukaryotes. Failure to ensure translation fidelity is often associated with stress related diseases and cancer.

The microbial biosphere represents the largest reservoir of catalysts for future industrial application. Yet it remains largely unexplored, with also the majority of genes identified in metagenomes still of unknown function. Using dehalogenation as a key major function with high-potential, versatile biocatalytic activity, dehalofluidX aims to address the major bottleneck faced by metagenomics today: the increasing recognition that genuinely novel biocatalysts with desired properties and unrelated sequences to already known enzymes, will only become accessible through novel approaches and pipelines. DehalofluidX is a tightly focussed 36-month innovative collaborative research project associating 3 partners from 3 research units in France, with complementary areas of expertise in microbial dehalogenation, microfluidics and omics technologies, and in particular microbial proteomics. The innovative concept of the high-throughput microfluidics-driven workflow is to use fluorescence quenching by halide ions in water-in-oil droplets to detect dehalogenation activity. This readout will be applicable to all halogenated compounds for which discovery of a novel dehalogenase catalyst is desirable. DehalofluidX does not require selective growth of dehalogenating organisms, and will target a much larger diversity of dehalogenating microbes and biocatalysts of the microbial biosphere than that accessed so far. DehalofluidX is organised in 3 successive well-defined, interconnected experimental tasks: (i) Droplet-based activity screening (validation of the microfluidics screening pipeline; isolating dehalogenating bacteria from environmental samples); (ii) Processing of cells with dehalogenase activity (processing of cultivable, poorly cultivable or non-cultivable cells with dehalogenase activity; phylogenetic and proteomic characterization, PCR screening for known dehalogenases); and (iii) Characterisation of novel catalysts (identification of genomic regions encoding dehalogenase activity; enzymatic characterisation of newly identified dehalogenases). Through its goal of discovering novel enzymes from the environmental microbiome for subsequent application and/or optimisation by biotech companies, dehalofluidX perfectly fits axis 3 of ANR Défi 3, as it may yield both new basic knowledge and technological know-how indispensable for the discovery of new biocatalysts. DehalofluidX also fits well with the National Strategy of Research ("green factory", Orientation 12 of the SNR), and has a direct link with the “Investments for the Future” program through partner DBR, member of LabEx NetRNA. The project PI has access to both pristine and contaminated environments as a source of new dehalogenating microbes and enzymes. Strong consortium expertise in all aspects of the proposed work strongly reduces potential challenges to success. Key requirements of droplet-based microfluidic high-throughput screening (droplet stability, maintenance of phenotype-genotype linkage, and signal sensitivity) have already been validated in preliminary experiments of halide-dependent fluorescence quenching in the presence of halogenated substrates. A thorough risk assessment was also performed on all stages of the dehalofluidX workflow, and potential issues and experimental fall-back solutions were identified and described. DehalofluidX will have major scientific impact, through delivery of novel biocatalysts for sustainable production of chemicals and bioremediation, and proof-of-concept of a novel activity-driven microfluidics high-throughput screening pipeline, expected to become generally applicable to other types of catalysts and R&D topics in the future. Technological impact will also be significant due to the novelty of the proposed activity-based approach for enzyme discovery. From an economic viewpoint, newly identified catalysts will be patented and licensed, as handled by SATT Conectus, the technology transfer instrument at Université de Strasbourg.

RNA modifications are involved in numerous biological processes and are present in all classes of RNA. These modifications are constitutive or modulated in response to adaptive processes and can impact RNA base pairing formation, protein recognition, RNA structure and stability. However, their roles in stress, environmental adaptation responses and infections caused by pathogenic bacteria, have just started to be appreciated. We make the assumption that RNA modifications in bacteria may be more ubiquitous and physiologically important than presently suspected. With the development of modern technologies in mass spectrometry and deep sequencing, and the possibility to test the impact of RNA modifications on the infection process, the three partners decided to join their forces and complementary expertise in order to determine the contribution of post-transcriptional modifications in non-coding and stable RNAs in translation and its regulation at a global scale in one of the major human pathogen, Staphylococcus aureus. The SaRNAmod project is based on three interconnected tasks and on a comparative study that will be performed on two specific strains, the methicillin-susceptible and genetically tractable HG001 strain and the methicillin-resistant USA300 clinical strain (i) We first aim at establishing the global profiles of post-transcriptional modifications in stable RNAs including tRNAs, ribosomal RNAs, and some selected regulatory RNAs acting as translational regulators (sRNA). This mapping will be done using state-of-the art methodologies including mass spectrometry (P1) and deep sequencing P2). Purification of specific RNAs for precise characterization of their modifications is available. (ii) We will analyze the dynamic-regulated modifications in tRNAs and rRNAs in response to specific stresses encountered during infection (i.e. oxidative and nitric oxide sensing) and to anti-toxinic antibiotic treatments. We will make use of sophisticated approaches such as ribosome profiling, to decipher the impact of modification deregulation on the decoding process. (iii) Using specific mutations at modifier enzymes, we will analyze their impact on S. aureus physiology and pathogenesis and relate the expression of specific modification enzymes with specific infections (P3). The outcomes of the project are expected to generate major breakthroughs: (i) at the technical level with the development of new methods of mass spectrometry and deep sequencing to map the set of modified bases of any RNA (particularly for the detection of pseudouridines); (ii) at the basic research level, with complete mapping of the modified bases of S. aureus non-coding RNAs, their functional impact on the physiology and antibiotic resistance mechanisms, and characterization of modification enzymes as well as proteins regulating the modifications of RNAs; (iii) at the medical level, the identification of new targets for the search for strategies to interfere with virulence and / or bacterial growth. The network lies in the combination of complementary expertise including RNA biology and deep mechanistic insights in the translation process, development of MS approaches linked to RNA biology (P1: S. Marzi), detection of RNA modifications by deep sequencing and biological functions of their machineries (P2: Y. Motorin), the study of staphylococcal pathogenesis, virulence and resistance to antibiotics (P3: F. Vandenesch). This synergy represents a unique opportunity to unite biochemical, genetics, structural, proteomics and transcriptomics skills, infection models and patient isolated strains, in a common effort to take up this challenge of determining the epitranscriptome and its regulation in this major human pathogen and to open new avenues for therapeutic applications.

Fundamental biological processes require metabolism adaptation mainly through translation regulation in order to respond to various stimuli ranging from environmental changes, stress responses or viral infections. These sophisticated processes are the result of the interplay between numerous actors: the ribosome, cis-acting elements located in ribosomal RNA or in mRNA called RNA regulons and trans-acting factors such as translation initiation factors. Therefore, assessing the complexity of translation regulation by in vivo approaches is a very difficult task and often leads to challenging interpretation. Consequently, there is a great demand for simplified in vitro approaches that will enable a better understanding of the molecular basis of these translation regulation mechanisms. The goal of this proposal is to answer to this demand and to develop a novel and innovative global in vitro method. For that purpose we will develop a novel microfluidic approach combining the use of fluorescent reporter-coding mRNA, cell-free translation extracts and Next Generation Sequencing. This method will enable fast fluorescence-based sorting of active/inactive mRNA variants from large libraries that will allow deciphering the molecular mechanisms of many translation regulation processes in a fast and systematic however global manner. Our consortium is composed of two partners, Franck Martin (coordinator) and Michael Ryckelynck who are experts in translation and microfluidic respectively. We already performed several seminal proof-of-concept experiments. Indeed, with this novel microfluidic approach, we investigated exhaustively the decoding rules in eukaryotic translation, which allowed us to determine all the codon-anticodon combinations that are readily accepted by the ribosome. This proof-of-concept experiment led to the discovery of remarkable fundamental aspects of eukaryotic translation, for example we found that anticodons GGN are detrimental to decoding fidelity. Interestingly, phylogenetic analysis showed that these anticodons do not exist in eukaryotes suggesting that they have been cleared in evolution to avoid misreading issues that were predicted by our selections. This striking result confirmed that our microfluidic approach is a novel powerful tool that can lead to fundamental discoveries in the translation field. In the frame of this proposal we will first exploit this new technology to study other new fundamental issues in eukaryotic translation. Recently, start codon selection has become a major issue in the translation field. We will address this topic with our microfluidic approach in order to determine the optimal start codon sequences and the impact of trans-acting factors (e.g. eIF5). We will also study non-AUG translation and determine exhaustively all the alternative codons that can efficiently support translation initiation with the eukaryotic ribosome. Then, we will further develop this approach to tackle other specific issues in the translation control field. The main goal will be to characterize novel RNA regulons that modulate (activate or inhibit) eukaryotic translation. This will require further developments of our methodology to be able to perform positive and negative selections. We will screen large RNA libraries in order to identify new RNA regulons such as Internal Ribosome Entry Sites, Translation Inhibitory Elements or Translation Enhancer Elements. As a proof-of-concept, we will focus on two (+) ssRNA viral genomes, Dengue and Sindbis for viral RNA regulons selections. Then we will use the same strategy to identify new RNA regulons in the human genome by screening human 5’ and 3’ UTR libraries. The last part of this project will be to extent our novel microfluidic approach to prokaryotic translation; the ultimate goal being to develop an efficient drug screening pipeline to discover translation-specific inhibitors leading to new antibiotic, a major issue for human health in the coming years.

Non-coding pervasive transcription initiating from cryptic signals or resulting from terminator read-through is widespread in all organisms. Its biological role is well-established in eukaryotes, but poorly understood in bacteria. Two major mechanisms control bacterial pervasive transcription: transcription termination by Rho and RNA degradation by RNases. Our recent data suggest a connection between these two pathways. The multidisciplinary project CoNoCo aims to define the mutual contributions of Rho and RNase III in the control of pervasive transcription in the Gram-positive model bacteria Bacillus subtilis and Staphyloccoccus aureus. It will also establish the roles of the non-coding transcriptome in bacterial cell biology highlighted by recent discoveries of Rho-mediated regulation of B. subtilis cell differentiation and the involvement of the double-strand specific RNase III in gene regulation by small non-coding RNAs.