Structure, function and evolution of armless mitochondrial tRNAs Tina Jühling To cite this version: Tina Jühling. Structure, function and evolution of armless mitochondrial tRNAs. Parasitology. Uni- versité de Strasbourg; Karl-Marx-Universität Leipzig, 2016. English. NNT : 2016STRAJ119. tel- 01673831v2 HAL Id: tel-01673831 https://tel.archives-ouvertes.fr/tel-01673831v2 Submitted on 11 Jan 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTÉ Architecture et Réactivité de l’ARN – UPR 9002 du CNRS et / und UNIVERSITÄT LEIPZIG FAKULTÄT FÜR BIOWISSENSCHAFTEN, PHARMAZIE UND PSYCHOLOGIE THÈSE / DISSERTATION présentée par / vorgelegt von Tina MÜLLER (ép. JÜHLING) soutenue le / verteidigt am 14.12.2016 pour obtenir le grade de Docteur de l’université de Strasbourg (Dr.) zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) Discipline/Spécialité : Aspects moléculaires et cellulaires de la biologie im Fachgebiet : Biochemie ARNt « manchots » structure, fonctionnalité et évolution THÈSE co-dirigée par: Prof. FLORENTZ Catherine Professeur, IBMC, Université de Strasbourg Prof. MÖRL Mario Professeur, Institut de Biochimie, Université de Leipzig RAPPORTEURS : Prof. LAYER Gunhild Professeur, Institut de Biochimie, Université de Leipzig Prof. CHIHADE Joseph Professeur, Department of Chemistry, Carleton College AUTRES MEMBRES DU JURY : Prof. GIEGÉ Philippe Directeur de recherche, IBMP, Université de Strasbourg Structure, function and evolution of armless mitochondrial tRNAs Der Fakultät für Biowissenschaften, Pharmazie und Psychologie der Universität Leipzig und / et École Doctorale des Sciences de la Vie et de la Santé Architecture et Réactivité de l’ARN – UPR 9002 du CNRS Université de Strasbourg eingereichte D I S S E R T A T I O N / T H È S E zur Erlangung des akademischen Grades / pour obtenir le grade de DOCTOR RERUM NATURALIUM (Dr. rer. nat.) und / et DOCTEUR DE L’UNIVERSITÉ DE STRASBOURG (Dr.) im Fachgebiet / Discipline-Specialité Biochemie / Aspects Moléculaire et Cellulaires de la Biologie vorgelegt von / présentée par Frau Master of Science Tina Jühling (geb. Müller) Geboren am / née le 25.07.1988 in Haldensleben Leipzig, den 10.10.2016 Le présent travail a été réalisé du 01/10/2013 au 30/04/2015 dans le groupe de Prof. Dr. Mario Mörl à l'Institut de biochimie, Faculté de Biosciences, pharmacie et de psychologie, Université de Leipzig, et du 01/05/2015 au 31/12/2016, dans le groupe de Prof. Dr. Catherine Florentz à l’IBMC/CNRS UPR9002 de l’Université de Strasbourg. Die vorliegende Arbeit wurde vom 01.10.2013 bis zum 30.04.2015 in der Arbeitsgruppe von Prof. Dr. Mario Mörl am Institut für Biochemie der Fakultät für Biowissenschaften, Pharmazie und Psychologie der Universität Leipzig, sowie vom 01.05.2015 bis zum 31.12.2016 in der Arbeitsgruppe von Prof. Dr. Catherine Florentz am IBMC/CNRS UPR9002 der Universität Strasbourg angefertigt. Nothing in life is to be feared, it is only to be understood. Marie Curie (7 November 1867 – 4 July 1934) Acknowledgments It is a great pleasure for me to thank all those who have directly or indirectly contributed to this work during the last three years. First of all, I want to thank the jury members Prof. Gunhild Layer (Institut für Biochmie, Universität Leipzig), Prof. Joseph Chihade (Carleton College, Northfield, Minnesota), and Prof. Philippe Giegé (IBMP, Université de Strasbourg), for their interest in my work and its evaluation. Foremost, I want to express my sincere gratitude to my two thesis directors Prof. Catherine Florentz and Prof. Mario Mörl for sharing the responsibility of my joint PhD project. I am very grateful for your trust and support over the past three years. I would like to thank you, Catherine, for the opportunity to join your group again, five years after I finished my Bachelor thesis in your team. Then as now, I spent a wonderful time in your group and had benefit a lot from your immense knowledge and guidance during my research and writing of this thesis. I also want to thank you, Mario, for the heartily admission in your team during my time in Leipzig. I appreciated very much the continuous support during the course of my PhD study as well as your patience, motivation, and enthusiasm for my project. I want to express my deepest gratitude to my advisor Dr. Joern Pütz, for his great support during my several stays at Strasbourg that started already during the French- German Bachelor program. Thanks to you, I spent a wonderful time as an Erasmus student in Strasbourg, and discovered the interesting world of tRNAs during my internship and Bachelor thesis. I thank you for all the help and support that you spent to establish this “Cotutelle de thèse”. I am very grateful that you always had an open ear for my little and big problems. Your comforting and encouraging words motivated me a lot. Thank you for all! I also acknowledge Dr. Heike Betat and Dr. Marie Sissler, for their conscientious guidance, support, valuable help, and for all the encouragements during the last three years. Thank you very much for answering my numerous questions, for your helpful advises and stimulating discussions. In addition, I would like to thank Prof. Jens Wöhnert, Dr. Elke Duchardt-Ferner, and Dr. Claude Sauter for their collaboration and help to determine the structures of armless tRNAs. I want to say “Danke”, “merci” and “thank you” to all my colleagues in Leipzig and Strasbourg. Thanks to you, working together in the laboratories has been a lot of fun. I will never forget our common barbecues, hiking trips, bocha games, and drinking evenings. Thank you Agnes, Anette, Bernard, Christian, Claude, Elena, Felix, Gesine, Karoline, Maja, Marie, Loukmane, Oliver, Oscar, Pablo, Paul, Raphael, Rebecca, Sandra, Sonja, Tobias, and Ulrike for a great atmosphere and a wonderful time. I want especially thank my parents Sigrid and Fritz Müller for their endless love and moral, and of course, for their financial support. You supported me always when I needed your help, consoled me when I was sad, and got excited when I was happy. To say it shortly, you have always been there for me. To my love Frank, I want to say that you delight my life. I am very grateful that you have accompanied me through the ups and downs of this work, and that you always stayed on my side. Without your contribution to the discovery of armless tRNAs, this thesis work might have not arisen. I am so glad to spend my life with you and love you wholeheartedly. Last but not least, I want to thank all my friends in Leipzig and Strasbourg, who directly or indirectly helped me to complete this thesis, especially Alexis for my daily “lab dance”, and Caro, Maja, Anne-So and Red for having enriched my life outside the lab. Bibliographic references and abstract Tina Jühling (née Müller) Structure, function and evolution of armless mitochondrial tRNAs Fakultät für Biowissenschaften, Pharmazie Architecture et Réactivité de l’ARN (ARN) und Psychologie IBMC/CNRS UPR 9002 Universität Leipzig Université de Strasbourg Thesis 202 pages, 336 references, 62 figures, 13 tables Transfer RNAs (tRNAs) are important adapter molecules linking the genetic information of messenger RNAs (mRNA) with the primary amino acid sequence of proteins. In all kingdoms of life, these small RNA transcripts have a typical cloverleaf-like secondary structure, consisting of an acceptor stem, a D-arm, an anticodon arm, a variable loop, and a T-arm. The 3′ terminus ends with the CCA sequence, which is often post- transcriptionally added by CCA-adding enzymes. The CCA-tail is an important prerequisite for the attachment of the correct amino acid by aminoacyl-tRNA synthetases (aaRS). Mitochondrial (mt) tRNAs show a high derivation from this canonical tRNA structure with reduced D- or T-arms, or even completely lack one of these elements. An extreme case of structural truncations can be observed in mitochondria of Enoplea. Here, mitochondrial tRNAs of half of the size of their cytosolic counterparts are present, representing the smallest tRNAs identified so far. It could already be shown that several of these miniaturized armless tRNAs are indeed functional in the nematode worm Romanomermis culicivorax. This situation raises several questions concerning the molecular mechanisms of co-evolution of tRNAs and their partner proteins, which ensure the maintenance of a functional protein synthesis. This study aims the biofunctional characterization of such “bizarre” tRNAs in defining their structural properties, and in studying different aspects of their functionality, especially their interactions with CCA-adding enzymes and aaRSs from different organisms. For this purpose, in vitro transcripts were used for structure probing approaches, such as enzymatic and chemical probing, nuclear magnetic resonance (NMR) spectroscopy, and small-angle X-ray scattering (SAXS). We show that armless tRNAs form a hairpin-shaped secondary structure, including an internal double bulge that replaces D- and T-arms of the secondary cloverleaf structure of classical tRNAs. 3D 1 structures are characterized by a high intrinsic flexibility, which probably allows to compensate structural reduction. The Rcu CCA-adding enzyme and mt ArgRS coding sequences have been identified, and were cloned for the first time. Recombinant proteins have been studied for their interaction with armless and cytosolic tRNAs in CCA-incorporations assays, and aminoacylation assays. We demonstrate that armless mt tRNAs represent functional molecules for CCA-incorporation, indicating adaptations of CCA-adding enzymes to armless tRNAs without losing their ability to recognize cytosolic tRNAs.