RiboFluidiX

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.