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Investigation of Cap-Independent Translation in Neuronal Differentiation

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Investigation of Cap-Independent Translation in Neuronal Differentiation Investigation of cap-independent translation in neuronal differentiation D I S S E R T A T I O N zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) eingereicht an der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin von M.Sc. Larissa Ruhe Präsidentin der Humboldt-Universität zu Berlin Prof. Dr.-Ing. Dr. Sabine Kunst Dekan der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin Prof. Dr. Bernhard Grimm Gutachter: 1. Prof. Dr. Nikolaus Rajewsky 2. Prof. Dr. Alexander Löwer 3. Ph.D. Marina Chekulaeva Tag der mündlichen Prüfung: 17.12.19 ABSTRACT Translation initiation is a complex and highly regulated process which involves the assembly of an elongation competent ribosome on the mRNA. The vast majority of eukaryotic mRNAs is translated by a canonical cap-dependent mechanism. This requires the eIF4F protein complex to bind the mRNA at the 5’-cap to recruit further eIFs and the small ribosomal subunit which then scans the 5’UTR in 5’ to 3’ direction until a start codon is encountered. Afterwards the large ribosomal subunit joins and protein synthesis begins. Besides that, translation of mRNAs can be mediated by IRESs, internal ribosome entry sites, which recruit the ribosome in a cap and 5’-end-independent manner to the start codon. Such cellular IRES-mediated translation is thought to be inefficient under physiological conditions but activated during stress. As the regulation of this mechanism is not well understood, we aimed to elucidate cellular cap-independent translation events. Therefore, we generated a mouse embryonic stem cell line with inducible overexpression of a dominant negative mutant of 4E-BP1. 4E-BP1 sequesters the cap-binding protein eIF4E so that the eIF4F protein complex fails to assemble at the 5’-cap. We performed shotgun proteomics during 4E-BP1 overexpression and neuronal differentiation to globally monitor translation dynamics. Genes with reduced sensitivity for cap-dependent translation were identified and tested for internal translation initiation in bicistronic reporter assays. After stringent validation one cap- independently translated mRNA, Pqbp1, was discovered. The second part of this study investigated cap-independent translation initiation on a circRNA, which by nature lacks free ends and thus requires IRES-mediated translation. We could show that circMbl is translated in vitro and thus contributed to the scientific evidence for the translation of circRNAs in fly brain, which was studied in a collaboration project. ZUSAMMENFASSUNG Initiation der Translation ist ein komplexer und stark regulierter Prozess, bei dem Ribosomen die mRNA binden. Die überwiegende Mehrheit eukaryotischer mRNAs wird durch einen 5‘-Cap- abhängigen Mechanismus translatiert. Dazu bindet der eIF4F-Proteinkomplex die mRNA an der 5'-Cap-Struktur, um weitere eIFs und die kleine ribosomale Untereinheit zu rekrutieren, welche dann die 5'UTR von 5'- in 3'-Richtung bis zu einem Startcodon scannt. Anschließend trifft die große ribosomale Untereinheit dazu und die Proteinsynthese beginnt. Darüber hinaus kann die Translation durch IRES, interne ribosomale Eintrittsstellen, vermittelt werden, welche das Ribosom unabhängig von Cap und 5‘-Ende zum Startcodon rekrutieren. Die zelluläre IRES-vermittelte Translation gilt als ineffizient unter physiologischen Bedingungen, wird aber durch Stress aktiviert. Da die Regulation dieses Mechanismus weitaus unbekannt ist, haben wir die zelluläre, Cap-unabhängige Translationsinitiation untersucht. Dafür haben wir eine embryonale Stammzelllinie generiert, welche eine dominant-negative Mutante von 4E-BP1 exprimiert. 4E-BP1 bindet das 5‘-Cap-bindende Protein, sodass eIF4F nicht am 5'-Cap andocken kann. Wir haben das Proteom während der Überexpression von 4E-BP1 und der neuronalen Differenzierung bestimmt, um Translationsdynamiken systemisch zu erfassen. Gene mit verminderter Sensitivität für die Cap-abhängige Translation wurden so identifiziert und in bicistronischen Reporter-Assays getestet. Nach strenger Validierung wurde eine Cap-unabhängig translatierte mRNA, Pqbp1, entdeckt. Der zweite Teil dieser Studie untersuchte die Cap-unabhängige Translation einer circRNA, welche keine freien Enden hat und daher per IRES translatiert werden muss. Wir konnten bestätigen, dass circMbl in vitro translatiert wird und konnten so innerhalb eines Kooperationsprojekts zu der Erkenntnis beitragen, dass circRNAs im Fliegengehirn translatiert werden. II SELBSTSTÄNDIGKEITSERKLÄRUNG Hiermit versichere ich, Larissa Ruhe, dass ich die vorliegende Dissertation selbstständig und auf der Grundlage der angegebenen Hilfsmittel und Hilfen angefertigt habe. Ich erkläre, dass ich sämtliche in der Arbeit verwendeten fremden Quellen und Grafiken als solche kenntlich gemacht habe. In Kooperation erhobene Daten und Ergebnisse wurden ebenfalls im Text als solche kenntlich gemacht. Dazu gehört die Probenaufbereitung, Durchführung und Datenaufarbeitung der massenspektrometrischen Analyse durch Dr. Guido Mastrobuoni. Die Experimente zu circMbl habe ich eigenständig durchgeführt, diese waren aber Teil eines Kollaborationsprojektes, welches von Prof. Dr. Sebastian Kadener initiiert wurde und dessen Ergebnisse in Zusammenarbeit mit Dr. Nagarjuna Reddy Pamudurti, Dr. Osnat Bartok, Dr. Marvin Jens, Dr. Reut Ashwal-Fluss, Christin Sünkel (Stottmeister), Dr. Mor Hanan, Dr. Emanuel Wyler, Dr. Daniel Perez-Hernandez, Evelyn Ramberger, Shlomo Sheniz, Moshe Samson, Dr. Gunnar Dittmar, Prof. Dr. Markus Landthaler, Dr. Marina Chekulaeva und Prof. Dr. Nikolaus Rajewsky entstanden und bei Molecular Cell publiziert wurden [1]. Diese Arbeit oder Teile davon wurden bei keiner anderen wissenschaftlichen Einrichtung eingereicht, angenommen oder abgelehnt. Ich besitze keinen Doktorgrad und habe mich an keiner anderen Stelle um einen Doktorgrad beworben. Die dem Promotionsverfahren zugrunde liegende Promotionsordnung habe ich zur Kenntnis genommen. Weiterhin erkläre ich, dass keine Zusammenarbeit mit gewerblichen Promotionsberaterinnen/-beratern stattgefunden hat und dass die Grundsätze der Humboldt-Universität zu Berlin zur Sicherung guter wissenschaftlicher Praxis eingehalten wurden. Berlin, 27.05.2019 III TABLE OF CONTENT ABSTRACT ...................................................................................................................................................... I ZUSAMMENFASSUNG ................................................................................................................................... II SELBSTSTÄNDIGKEITSERKLÄRUNG ......................................................................................................... III TABLE OF CONTENT .................................................................................................................................. IV LIST OF FIGURES ........................................................................................................................................ VI LIST OF TABLES ......................................................................................................................................... VII ABBREVIATIONS ........................................................................................................................................ VII 1 INTRODUCTION ....................................................................................................................................... 21 1.1 The canonical mechanism of translation initiation in eukaryotes: 5’end-dependent initiation ................................................................................................................................................................... 21 1.2 Noncanonical mechanisms of translation initiation: cap-independent and internal initiation ................................................................................................................................................................... 23 1.2.1 Translation initiation by internal ribosome entry sites (IRESs) in viruses ....................... 23 1.2.2 Translation initiation by internal ribosome entry sites (IRESs) in human ....................... 25 1.2.3 Cap-independent translation initiation at the 5’end: CITE-like mechanisms .................. 41 1.3 Global and gene-specific translational regulation ....................................................................... 46 1.3.1 Molecular properties of the translational repressor eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) ............................................................................................ 47 1.3.2 Biological functions of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) .............................................................................................................................................. 49 1.3.3 RNA binding proteins and sequence elements in untranslated regions ........................... 50 1.3.4 m6A mRNA methylation ......................................................................................................... 53 1.3.5 Upstream open reading frames (uORFs) and start codons (uAUGs) .............................. 54 1.3.6 miRNA binding sites ................................................................................................................ 58 1.3.7 mRNA secondary structure .................................................................................................... 61 1.4 Translational control in physiological and
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