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The Functions of Y-Box Binding Proteins in Caenorhabditis Elegans

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The Functions of Y-Box Binding Proteins in Caenorhabditis Elegans THE FUNCTIONS OF Y-BOX BINDING PROTEINS IN CAENORHABDITIS ELEGANS Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Andreas Arnold Aus der Schweiz Basel, 2015 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Susan M. Gasser Prof. Dr. Raúl Méndez Dr. Rafal Ciosk Basel, 24.03.2015 Dekan: Prof. Dr. Jörg Schibler [2] TABLE OF CONTENT ABBREVIATIONS ...................................................................................................... 5 1. SUMMARY ............................................................................................................. 7 2. GENERAL INTRODUCTION .................................................................................. 8 2.1 The multiple layers of gene regulation ............................................................................ 8 2.2 The mature messenger RNA molecule ......................................................................... 10 2.3 The ribosome ................................................................................................................ 12 2.3.1 Ribosome biogenesis ............................................................................................. 12 2.3.2 Ribosome maturation and quality control ............................................................... 12 2.3.3 Ribosome heterogeneity ........................................................................................ 14 2.4 Translating mRNA ........................................................................................................ 15 2.4.1 Translation initiation ............................................................................................... 15 2.4.2 Translation elongation and termination .................................................................. 17 2.5 Degrading mRNA .......................................................................................................... 19 2.5.1 Degradation mechanisms ...................................................................................... 19 2.5.2 Translation versus mRNA degradation .................................................................. 20 2.5.3 Promoting the repression/degradation of “healthy” mRNA .................................... 21 2.5.4 Degradation of aberrant mRNA .............................................................................. 22 2.5.5 Processing bodies and stress granules ................................................................. 24 2.6 Storing mRNA ............................................................................................................... 25 2.6.1 Storing maternal mRNA in the Xenopus oocyte ..................................................... 26 2.6.2 Storing maternal mRNA in the Drosophila oocyte .................................................. 27 2.7 The importance of general RNA-binding proteins in PTGR .......................................... 28 2.8 The family of Y-box binding proteins ............................................................................ 29 2.8.1 YB-1: A paradigm for pleiotropic functionality ........................................................ 29 2.8.2 The role of Y-box binding proteins in maternal mRNA regulation .......................... 32 2.9 The nematode Caenorhabditis elegans ........................................................................ 33 [3] 2.9.1 The reproductive system ........................................................................................ 34 2.9.2 GLD-1 and CGH-1: Two essential RNA-binding proteins in oogenesis ................. 36 3. SCOPE OF THIS PHD THESIS ............................................................................38 4. RESULTS ..............................................................................................................39 4.1 Functional characterization of C. elegans Y-box binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes ................................................. 39 4.1.1 Abstract .................................................................................................................. 40 4.1.2 Introduction ............................................................................................................ 40 4.1.3 Materials and methods ........................................................................................... 41 4.1.4 Results ................................................................................................................... 48 4.1.5 Discussion .............................................................................................................. 56 4.1.6 Funding .................................................................................................................. 58 4.1.7 Acknowledgements ................................................................................................ 59 4.1.8 References ............................................................................................................. 59 4.1.9 Figures ................................................................................................................... 63 4.1.10 Supplementary figures and tables ........................................................................ 76 4.2 RG/RGG repeats regulate the abundance of CEY proteins ....................................... 103 4.2.1 Introduction .......................................................................................................... 103 4.2.2 Materials and Methods ......................................................................................... 103 4.2.3 Results and discussion ........................................................................................ 103 5. GENERAL DISCUSSION AND OUTLOOK ........................................................106 5.1 Functions of CEY proteins in the germline ................................................................. 106 5.2 Functions of CEY proteins in the soma ...................................................................... 108 6. ACKNOWLEDGEMENTS ...................................................................................113 7. REFERENCES ....................................................................................................114 8. APPENDICES .....................................................................................................129 [4] ABBREVIATIONS A site Acceptor site ADMA Asymmetrical dimethylation AF Assembly factor ARE AU-rich element APA Alternative polyadenylation CEY C. elegans Y-box binding protein CDS Coding sequenc CPE Cytoplasmic polyadenylation element CPEB Cytoplasmic polyadenylation element binding protein CSD Cold shock domain Csp Cold shock protein CTD C-terminal domain DTC Distal tip cell E site Exit site eEF Eukaryotic translation elongation factor eEF2K eEF2 kinase eIF Eukaryotic translation initiation factor IRES Internal ribosome entry site miRISC miRNA-induced silencing complex miRNA micro RNA mRNA messenger RNA mRNP messenger ribonucleoparticle NGD No-go decay NMD Nonsense-mediated decay NRD Non-functional rRNA decay NSD Non-stop decay P bodies Processing bodies P site Peptidyl-tRNA site PABP Poly(A) binding protein PIC Preinitiation complex PTC Premature termination codon PTGR Post-transcriptional gene regulation RBP RNA binding protein RNAi RNA interference [5] RNA pol RNA polymerase RPF Ribosome protected fragment rRNA Ribosomal RNA SUnSET Surface sensing of translation TGR Transcriptional gene regulation tRNA transfer RNA UTR Untranslated region YBP Y-box binding protein [6] 1. SUMMARY Members of the highly conserved family of Y-box binding proteins (YBPs) have a broad spectrum of functions in both transcriptional and post-transcriptional regulation of gene expression. However, most information comes from in vitro or single cell experimental systems. In addition, these proteins have not been studied in one of the major model organisms, Caenorhabditis elegans. Here, we provide a functional characterization of YBPs in this nematode, thereby also generally adding to the very scarce knowledge on the developmental functions of YBPs in a multicellular context. Our data suggests a conserved and essential role for C. elegans YBPs (CEYs) in packaging and stabilizing maternal mRNAs during oogenesis. In the soma, the absence of CEYs also affects the abundance of many messages, interestingly, with a strong bias for highly translated mRNAs. The most striking observation is the soma-specific requirement for CEY proteins to accumulate large polysomes. Surprisingly, this loss has no negative impact on translation rates and cey mutant animals show no apparent defects in somatic development. Finally, our data also suggests a potentially novel function for YBPs, namely, a direct involvement in ribosome biology. [7] 2. GENERAL INTRODUCTION 2.1 The multiple layers of gene regulation The “building plan” for any organism is encoded in its DNA, more precisely, by a large array of individual modules, so called genes. Correct temporal and spatial regulation of gene expression is essential from a developmental point of view, but also as a mean to respond to changes in environmental conditions. In eukaryotes, the DNA is located in the nucleus, which is separated by the nuclear
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