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Interactions and functions of RNA-binding proteins Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program Molecular biology of cells of the Georg-August University School of Science (GAUSS) submitted by Jens Kretschmer from Frankfurt am Main Göttingen 2016 Thesis Committee Prof. Dr. Markus Bohnsack Department of Molecular Biology, University Medical Centre Prof. Dr. Peter Rehling Department of Cellular Biochemistry, University Medical Centre Prof. Dr. Heinz Neumann Max-Planck-Institute for Molecular Physiology, Dortmund Members of the Examination Board Referee: Prof. Dr. Markus Bohnsack Department of Molecular Biology, University Medical Centre 2nd Referee: Prof. Dr. Peter Rehling Department of Cellular Biochemistry, University Medical Centre Further members of the Examination Board Prof. Dr. Heinz Neumann Max-Planck-Institute for Molecular Physiology, Dortmund Prof. Dr. Claudia Höbartner Institute for Organic and Biomolecular Chemistry Prof. Dr. Ralf Ficner Dept. of Molecular Structural Biology, Institute for Microbiology and Genetics Prof. Dr. Jörg Stülke Dept. of General Microbiology, Institute for Microbiology and Genetics Date of oral examination: 20.01.2017 Erklärung Promovierenden-Erklärung Georg-August-Universität Göttingen Name: Kretschmer,Jens (Name, Vorname) Anschrift: Südring 19B, 37120, Bovenden (Straße, PLZ, Wohnort) Ich beabsichtige, eine Dissertation zum Thema “Interactions and functions of RNA- binding proteins” an der Georg-August-Universität Göttingen anzufertigen. Dabei werde ich von Herrn Prof. Dr. Markus T. Bohnsack betreut. Ich gebe folgende Erklärung ab: 1. Die Gelegenheit zum vorliegenden Promotionsvorhaben ist mir nicht kommerziell vermittelt worden. Insbesondere habe ich keine Organisation eingeschaltet, die gegen Entgelt Betreuerinnen und Betreuer für die Anfertigung von Dissertationen sucht oder die mir obliegenden Pflichten hinsichtlich der Prüfungsleistungen für mich ganz oder teilweise erledigt. 2. Hilfe Dritter wurde bis jetzt und wird auch künftig nur in wissenschaftlich vertretbarem und prüfungsrechtlich zulässigem Ausmaß in Anspruch genommen. Insbesondere werden alle Teile der Dissertation selbst angefertigt; unzulässige fremde Hilfe habe ich dazu weder unentgeltlich noch entgeltlich entgegengenommen und werde dies auch zukünftig so halten. 3. Die Richtlinien zur Sicherung der guten wissenschaftlichen Praxis an der Universität Göttingen werden von mir beachtet. 4. Eine entsprechende Promotion wurde an keiner anderen Hochschule im In- oder Ausland beantragt; die eingereichte Dissertation oder Teile von ihr wurden nicht für ein anderes Promotionsvorhaben verwendet. Mir ist bekannt, dass unrichtige Angaben die Zulassung zur Promotion ausschließen bzw. später zum Verfahrensabbruch oder zur Rücknahme des erlangten Grades führen. Göttingen, den 29.11.2016 (Unterschrift) Index Table of Contents Table of contents ..................................................................................................... I List of Figures ........................................................................................................ III List of Tables ........................................................................................................ IV Abbreviations ........................................................................................................ V Abstract ................................................................................................................ VI 1 Introduction ........................................................................................................ 1 1.1 RNA modifications ................................................................................................. 1 1.2 tRNA modifications ................................................................................................ 1 1.2.1 tRNA biogenesis and function ........................................................................ 1 1.2.2 Different types of tRNA modifications and their function ................................ 3 1.3 Ribosomal RNA modifications ............................................................................... 5 1.3.1 Biogenesis and function of ribosomes ............................................................ 5 1.3.2 snoRNA-guided modifications ........................................................................ 7 1.3.3 Base modifications ......................................................................................... 8 1.3.4 Functions of rRNA modifications .................................................................. 10 1.4 Messenger RNA modifications ............................................................................ 11 1.5 N6-methyladenosine ............................................................................................ 14 1.5.1 m6A in mRNA ............................................................................................... 14 1.5.2 m6A methyltransferases ............................................................................... 16 1.5.3 Oxidative demethylation ............................................................................... 17 1.6 Recognition of RNA modifications ....................................................................... 18 1.6.1 The YTH domain and m6A recognising proteins .......................................... 18 1.6.2 Functions of YTH domain proteins ............................................................... 20 1.6.3 Other m6A modification readers ................................................................... 22 1.7 Aims .................................................................................................................... 24 2 Materials and Methods ..................................................................................... 25 2.1 Materials .............................................................................................................. 25 2.1.1 Chemicals and enzymes .............................................................................. 25 2.1.2 Oligonucleotides ........................................................................................... 25 2.1.3 Plasmids used in this study .......................................................................... 27 2.1.4 siRNAs used in this study ............................................................................. 27 2.1.5 Antibodies used in this study ........................................................................ 28 I Index 2.2 Methods ............................................................................................................... 28 2.2.1 Molecular cloning ......................................................................................... 28 2.2.2 Site-directed mutagenesis ............................................................................ 29 2.2.3 SDS-PAGE and western blotting .................................................................. 29 2.2.4 Cell culture ................................................................................................... 30 2.2.5 Generation of HEK293 stable cell lines ........................................................ 30 2.2.6 Immunofluorescence .................................................................................... 31 2.2.7 Immunoprecipitation ..................................................................................... 31 2.2.8 Pre-ribosome and sucrose density gradients and polysome profiling .......... 32 2.2.9 RNA interference .......................................................................................... 33 2.2.10 RNA extraction ........................................................................................... 33 2.2.11 Quantitative real time PCR ......................................................................... 34 2.2.12 Agarose-glyoxal gel electrophoresis and northern blotting ........................ 34 2.2.13 Pulse-chase labelling of RNA ..................................................................... 35 2.2.14 Cross-linking and analysis of cDNA (CRAC) .............................................. 35 2.2.15 Genome-wide mapping of deep sequencing data ...................................... 38 2.2.16 Recombinant expression of proteins in E. coli ............................................ 39 2.2.17 Purification of His-tagged proteins ............................................................. 39 2.2.18 Anisotropy .................................................................................................. 40 3 Results ............................................................................................................. 41 3.1 Bioinformatic analysis of high throughput next-generation sequencing data ..................................................................................................................... 41 3.1.1 Verification of the pipeline ............................................................................ 45 3.2 Identification of RNA interactions of the YTH domain-containing proteins .......... 47 3.3 YTHDC2 associates with ribosomal complexes .................................................. 55 3.4 The YTH domain of YTHDC2 recognises the 18S m6A in vitro ........................... 59 3.5 Analysis of the cellular function of YTHDC2 ........................................................ 64 4 Discussion .......................................................................................................
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