Mitochondrial and cell cycle functions of SLIMP Albert Antolin Fontes ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX (www.tdx.cat) i a través del Dipòsit Digital de la UB (diposit.ub.edu) ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX ni al Dipòsit Digital de la UB. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX o al Dipòsit Digital de la UB (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. En la utilització o cita de parts de la tesi és obligat indicar el nom de la persona autora. ADVERTENCIA. 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UNIVERSITAT DE BARCELONA FACULTAT DE FARMÀCIA I CIÈNCIES DE L’ALIMENTACIÓ Programa de doctorat en Biomedicina INSTITUTE FOR RESEARCH IN BIOMEDICINE MITOCHONDRIAL AND CELL CYCLE FUNCTIONS OF SLIMP Memòria presentada per Albert Antolin Fontes per optar al títol de doctor per la Universitat de Barcelona Lluís Ribas de Pouplana Albert Antolin Fontes Antonio Zorzano Olarte Thesis director PhD candidate Tutor Albert Antolin Fontes 2018 “It is the long history of humankind that those who learned to collaborate have prevailed” Charles Darwin TABLE OF CONTENTS SUMMARY ............................................................................................................. 1 RESUM .................................................................................................................... 3 ABBREVIATIONS ............................................................................................... 5 INTRODUCTION ................................................................................................ 9 DNA replication ............................................................................................... 11 Transcription ..................................................................................................... 14 Eukaryotic translation ...................................................................................... 16 Mitochondria ..................................................................................................... 19 Mitochondrial DNA .................................................................................... 21 Mitochondrial DNA expression and maintenance ................................. 22 Mitochondrial DNA replication ................................................................. 26 Mitochondrial translation ............................................................................ 28 Mitochondrial protein folding and degradation ...................................... 30 Aminoacyl tRNA synthetases ......................................................................... 33 The Seryl-tRNA Synthetase ........................................................................ 35 Initial characterization of SLIMP .............................................................. 37 Cell cycle ............................................................................................................ 40 OBJECTIVES ....................................................................................................... 45 MATERIALS AND METHODS ..................................................................... 49 RESULTS .............................................................................................................. 71 Chapter 1: SLIMP localization and cellular environment .......................... 73 1.1 SLIMP contains a mitochondrial signal peptide ............................... 73 1.2 SLIMP is localized in the mitochondrial RNA granules .................. 76 Chapter 2: Mitochondrial function of SLIMP/ SerRS2 heterodimer ...... 81 2.1 Mitochondrial SLIMP and SerRS2 protein levels are interdependent ......................................................................................................................... 81 2.2 SLIMP/SerRS2 heterodimer is essential for mitochondrial protein translation ...................................................................................................... 84 2.3 SLIMP/SerRS2 heterodimer is required for mitochondrial respiration ...................................................................................................... 85 2.4 SLIMP/SerRS2 depletion does not increase ROS production in S2 cell mitochondria .......................................................................................... 89 Chapter 3: SLIMP and LON protease interaction represses mtDNA replication .......................................................................................................... 91 3.1 SLIMP interacts with the substrate binding domain of LON protease .......................................................................................................... 91 3.2 SLIMP represses TFAM degradation by LON protease ................. 93 3.3 LON general mitochondrial protease activity is not affected by SLIMP ............................................................................................................ 95 Chapter 4: SLIMP controls cell cycle progression ...................................... 99 4.1 SLIMP is required for cell cycle progression ..................................... 99 4.2 SLIMP-depleted cells accumulate in G2 cell cycle phase .............. 101 4.3 G1 to S cell cycle progression is repressed by SLIMP ................... 107 4.4 SLIMP is not differentially expressed during cell cycle ................. 109 4.5 SLIMP depletion induces gene transcription and G2/M checkpoint ....................................................................................................................... 110 4.6 Cdc6 and ORC2 protein levels increase upon SLIMP knockdown ....................................................................................................................... 114 4.7 SLIMP depletion does not trigger apparent DNA damage .......... 115 Chapter 5: SLIMP in human cells ................................................................ 118 DISCUSSION ..................................................................................................... 121 CONCLUSIONS ............................................................................................... 137 SUPPLEMENTARY MATERIAL ................................................................. 141 REFERENCES .................................................................................................. 149 ACKNOWLEDGMENTS ............................................................................... 169 SUMMARY The mitochondrial Seryl-tRNA Synthetase (SerRS2) is a member of the class II tRNA synthetase family. The mature enzyme catalyses the ligation of serine to tRNASer in mitochondria. During the process of constructing a model for human disorders caused by mitochondrial tRNA aminoacylation deficiencies in Drosophila melanogaster, a previously uncharacterized paralogue of SerRS2 named Seryl-tRNA synthetase-Like Insect Mitochondrial Protein (SLIMP) was identified. SLIMP is a new type of aminoacyl tRNA synthetase-like protein that has acquired an essential function in insects. This fast evolving paralogue is a mitochondrial RNA-binding protein which lacks tRNA aminoacylation activity. It has been previously demonstrated that mitochondrial SLIMP interacts with its homologue SerRS2 and also with LON protease. We confirmed these interactions and we described the function of SLIMP by depleting its protein levels in Drosophila melanogaster S2 cells, which led to severe defects in mitochondrial function and cell cycle arrest.