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Use of High-Throughput Sequencing for the Characterization of Extracellular RNA and to Study the Dynamics of Bacterial RNA Modification

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Use of High-Throughput Sequencing for the Characterization of Extracellular RNA and to Study the Dynamics of Bacterial RNA Modification AVERTISSEMENT Ce document est le fruit d'un long travail approuvé par le jury de soutenance et mis à disposition de l'ensemble de la communauté universitaire élargie. Il est soumis à la propriété intellectuelle de l'auteur. Ceci implique une obligation de citation et de référencement lors de l’utilisation de ce document. D'autre part, toute contrefaçon, plagiat, reproduction illicite encourt une poursuite pénale. Contact : [email protected] LIENS Code de la Propriété Intellectuelle. articles L 122. 4 Code de la Propriété Intellectuelle. articles L 335.2- L 335.10 http://www.cfcopies.com/V2/leg/leg_droi.php http://www.culture.gouv.fr/culture/infos-pratiques/droits/protection.htm Ecole Doctorale BioSE (Biologie-Santé-Environnement) Thèse Présentée et soutenue publiquement pour l’obtention du titre de DOCTEUR DE l’UNIVERSITE DE LORRAINE Mention : « Sciences de la Vie et de la Santé » par Adeline GALVANIN Use of high-throughput sequencing for the characterization of extracellular RNA and to study the dynamics of bacterial RNA modification Le 17 septembre 2019 Membres du jury : Rapporteurs : Mme Magali FRUGIER Directrice de recherche CNRS Strasbourg Mme Sophie ROME Directrice de recherche INSERM Lyon Examinateurs : Mr David COELHO Chargé de recherche INSERM Nancy Mme Jessica GOBBO Chercheuse clinicienne CGFL Dijon Mr Rolf POSTINA Privatdozent JGU Mainz Mme Carine TISNE Directrice de recherche CNRS/Université Paris Diderot Paris « « Mr Mark Helm Professeur JGU, co-directeur de thèse Mr Iouri Motorine Professeur UL Nancy, co-directeur de thèse ----------------------------------------------------------------------------------------------------------------------------- ------ UMR 7365 CNRS-UL, Laboratoire IMoPA (Ingénierie Moléculaire et Physiopathologie articulaire) JGU (Johannes Gutenberg Universität) – Institut für Pharmazie und Biochemie Foreword: RNA are molecules that play roles far more important than a simple transfer of information from the head: the sacred stored genetic material to the working-class proteins. Indeed, RNA are not only the molecules which make the link between DNA and proteins, RNA are even in the centre, we can consider them at the wise of the cell. At the beginning of the life on Earth, they were there, pioneers, having catalytic activities before bequeathing them to enzyme proteins. At this time in the evolution, it was what we call “the RNA world” where these nucleic acids were ruling the whole cell, they were at the base for everything. Now, a lot of other molecular components took over but RNA are still strongly present in the cell and are involved in a multitude of essential functions. They have so much properties and attributions that it is impossible to list them in an exhaustive manner. If we add a layer of complexity and add RNA modification, splicing event in the equation, it becomes completely crazy. This is why molecular biology and especially the study of RNA are a huge field in fundamental research where we never stop discovering new things. In a world where technology flows in our veins, where high throughput techniques allowed to make progress science, I am glad to contribute to this vast domain by writing my phD thesis. “Our world is built on biology and once we begin to understand it, it then becomes a technology.” Ryan Bethencourt Acknowledgement / Remerciements: First of all, I would like to thank my director of thesis Yuri Motorin, for accepting me in master 1 internship and to have trusted in me since for master thesis and phD thesis. You gave me the opportunity to play a role in science. I thank you for your precious advises and the great supervision. I thank my co-director of thesis Mark Helm. Thank for having welcomed me in your laboratory and supervising me during these periods. I could discover new and interesting methods for RNA analysis, another team, another culture! I would like to thank all the members of the jury of my phD defense: Carine Tisne, David Coelho, Jessica Gobbo, Magali Frugier, Rolf Postina and Sophie Rome for your future important advises. I thank Stefano Marzi and David Coelho for being in my thesis advisory committee and for giving me good clues for the redaction and defense of my thesis. I thank all the partners involved in my phD project and especially Alexander Dalpke and his team for the close collaboration we initiated together. J’aimerais également remercier chaleureusement l’équipe ARN et de manière générale le Biopôle. J’avoue que la couleur verte pomme des murs ne va peut-être pas me manquer mais les personnes les côtoyant tous les jours c’est tout à fait différent. C’était un réel plaisir de travailler avec vous, de pouvoir compter sur beaucoup de personnes pour des questions de manips ou pas ! Je remercie la plateforme de séquençage UMS IBSLor 2008 dirigée par mon directeur de thèse Yuri Motorin et gérée par Virginie Marchand. Partie intégrante de mon projet de thèse, je me suis familiarisée et formée sur le séquençage à haut débit grâce à vous. J’aimerais également remercie Emilie qui m’a beaucoup soutenue sur la partie vésicules extracellulaires. Des échanges enrichissants au point de vue scientifique mais pas uniquement. J’aimerais remercier mes chères collègues Lilia et Valérie, toujours de bonne humeur et prêtes à m’aider quand j’en avais besoin. Lilia, ma voisine de bureau, merci pour tout le soutien que tu m’as apporté. Ensemble on se redonnait du baume au cœur. Merci à mes amis thésards qui comprennent tant cette magnifique galère qu’est la thèse. Merci pour les moments de détente nécessaires aux pauses midi, en soirée ou autour d’un morceau de gâteau. Merci à ceux qui ont déjà terminé : Arnaud, Madhia, Yoann, Gabriel, Benjamin et à ceux qui seront bientôt libérés : Liza, Timothé, Laeya, Fanny, Paul, Florent, Jean-Malo, Charbel … Ich möchte mich bei der ganzen Mannschaft aus Mainz bedanken. Thank you for having welcomed me in the team and make increase the proportion of French members. A strong thank for Christina and Dominik, you taught me a lot of things and helped for many questions. I wish good luck to Larissa, you have to continue on these cursed tRNA isolation stuffs. Of course, I will not forget to thank Stephan, Florian, Kaouthar and Aurellia. It was a great pleasure to be with you for these few months. I made real friends and hope we will continue to see each other after! PS: je suis tellement dégoutée d’avoir cassé la boule à neige de Mainz … Merci à toi Géraud, ensemble depuis le M1 et en « concurrence » pour la thèse. Au final, on s’est tous les deux retrouvés en thèse. Merci pour ton humour si particulier et pour toutes nos discussions sur la vie future. J’espère qu’on trouvera notre voie et qu’on sera épanouis. Je te remercie Dafné, ensemble dans la même galère depuis le DUT ! On a beaucoup partagé pendant toutes ces années, nos joies, nos coups de mou, nos pétages de câble, nos excitations (qui parfois retombaient…), enfin tout quoi. Merci d’avoir été et d’être là. Petite mais énergique ! Je tiens à remercier ma famille et mes amis qui même s’ils n’ont toujours pas vraiment compris ce que je faisais, m’ont toujours soutenu dans mes projets d’étude et professionnels. Merci à mes parents, j’espère que vous serez fiers de moi. Merci notamment à Laurélia, nos rendez- vous du mercredi m’ont permis de tenir moralement dans des moments pas toujours faciles. Je ne suis pas médecin mais ça ne t’empêche pas de m’appeler pour tous tes petits bobos ^^. Et enfin, à celui qui a dû me supporter au quotidien pendant ces trois années, qui m’a réconforté quand il le fallait, qui a su faire abstraction quand je stressais pour rien : « comme d’habitude ». Tu as été d’un soutien inébranlable, je ne suis pas sûre que j’aurais supporté cette étape de ma vie aussi bien sans toi. Merci à toi Loic… Table of content: Foreword: ....................................................................................................................................... Acknowledgement / Remerciements: ............................................................................................. Table of content: ............................................................................................................................. Scientific achievements: ................................................................................................................. Figure list: ...................................................................................................................................... Table list: ........................................................................................................................................ Abbreviation list: ............................................................................................................................ General introduction ................................................................................................................. I. Ribonucleic acids: RNA ....................................................................................................................... 1 II. Nucleic acids sequencing from 60s to now and later ........................................................................... 1 a. 1st Generation .................................................................................................................................. 2 b. 2nd Generation .................................................................................................................................. 3 c. 3rd Generation: Pacific
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