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Characterizing the Role of RNA-Binding Proteins in Ubiquitin

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Characterizing the Role of RNA-Binding Proteins in Ubiquitin CHARACTERIZING THE ROLE OF RNA-BINDING PROTEINS IN UBIQUITIN SIGNALING Dissertation zur Erlangung des Grades Doktor der Naturwissenschaften am Fachbereich Biologie der Johannes Gutenberg-Universität in Mainz Andrea Hildebrandt, M.Sc. Mainz, Juni 2019 1 Dekan: 1. Berichterstatter: 2. Berichterstatter: Tag der mündlichen Prüfung: Preface PREFACE Results of the presented work have partly been published previously in the following publications Chapter I.3 Sutandy FXR, Hildebrandt A, and König J. Profiling the Binding Sites of RNA- Binding Proteins with Nucleotide Resolution Using iCLIP. Methods Mol Biol. 2016. Chapter III Hildebrandt A et al. Interaction profiling of RNA-binding ubiquitin ligases reveals a link between posttranscriptional regulation and the ubiquitin system. Sci Rep. 2017. Chapter IV Hildebrandt A et al. 2019. The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A)-translation. bioRxiv; doi: 10.1101/516005 The work included in this thesis was conducted within a joint research project in the laboratories of Dr. Julian König and Dr. Petra Beli at the IMB in Mainz. Dr. Julian König and Dr. Petra Beli conceived and supervised this study. Moreover, this work was supported by research collaborations with the laboratories of Dr. Kathi Zarnack (BMLS, Frankfurt), Prof. Miguel A. Andrade-Navarro (JGU, Mainz), Dr. Jean-Yves Roignant, and Prof. René Ketting (both IMB, Mainz). I was involved in the design and evaluation of all experiments and analyses. All experimental work and data analyses were performed by myself, with the following exceptions: Experiments in the context of the publication Sutandy FXR, Hildebrandt, and König, 2016 (Chapter I.3), were performed together with Dr. FX Reymond Sutandy. The manuscript was written together with Dr. Julian König and Dr. FX Reymond Sutandy. In the context of the publication Hildebrandt et al. 2017 (Chapter III), Prof. Miguel A. Andrade-Navarro and Dr. Gregorio Alanis-Lobato (JGU, Mainz) developed a method for the evaluation of the adapted AP. Dr. Gregorio Alanis-Lobato performed computational functional analyses, including GO term analyses, WI determination, Precision-Recall analyses, and comparisons of detected interactions to the HIPPIE database. Dr. Kathi Zarnack was involved in the design and analysis of the study, contributed ideas and wrote the manuscript together with me. III Preface In the context of data presented in Hildebrandt et al. 2019 (Chapter IV), Dr. Anke Busch (IMB, Mainz), performed inital analyses of ubiquitylome and interactome data as well initial iCLIP data processing and analysis steps. Susan Boerner (BMLS, Frankfurt) also performed initial analyses of iCLIP data. Mirko Brüggemann (BMLS, Frankfurt) performed bioinformatics analyses of the MKRN1 iCLIP data. Cornelia Rücklée (BMLS, Frankfurt) analyzed MKRN1 binding at polyadenylation sites and poly(A) tails. Moreover, she contributed the evolutionary characterization of Makorin proteins under the supervision of Prof. Ingo Ebersberger (Goethe Universität, Frankfurt). Jan Heidelberger (IMB, Mainz) performed replicate ubiquitin remnant profiling experiments. Andrea Voigt (IMB, Mainz) performed AP-Western blot experiments and Western blot validations. Heike Hänel (IMB, Mainz) performed replicate iCLIP and replicate AP- Western blot experiments. Dr. Stefanie Ebersberger (IMB, Mainz) and Dr. Kathi Zarnack supervised the bioinformatics analyses. Moreover, Dr. Kathi Zarnack was involved in the design and analysis of the study. Annabelle Dold and Dr. Jean-Yves Roignant (both IMB, Mainz) performed complementary studies in D. melanogaster. The manuscript was written by myself, Dr. Kathi Zarnack, Dr. Julian König, and Dr. Petra Beli. Prof. René Ketting, Dr. Nadine Wittkopp, and Miguel V. Almeida participated in fruitful discussions. For Chapter V, Dr. Anke Busch analyzed the RNA-Seq and Pulsed SILAC data. Dr. Kathi Zarnack, Dr. Stefanie Ebersberger, and Susan Boerner were involved in the RNA- Seq analyses. Sonja Debeç (Summer student at IMB, Mainz) performed the final steps of the TOPO cloning protocol for pGL3pro-YWHAB and set up the experimental frame work for the Luciferase reporter assay. Heike Hänel helped with Western blots to characterize the cell lines stably expressing RPS10 (mutants). The IMB Genomics Core Facility prepared RNA-Seq libraries from submitted mRNA and sequenced all RNA-Seq and iCLIP libraries. Supervisor confirmation _______________________________ IV Content CONTENT PREFACE III CONTENT V ZUSAMMENFASSUNG VIII SUMMARY IX I. INTRODUCTION 12 1. BIRTH OF A FUNCTIONAL PROTEIN 12 1.1 RNA-BINDING PROTEINS 12 1.2 MRNA 3’END PROCESSING AND THE POLY(A) TAIL 16 1.3 POLY(A)-BINDING PROTEINS 18 1.4 RIBOSOMES 18 1.5 TRANSLATION 19 1.6 OBSTACLES IN TRANSLATION AND CO-TRANSLATIONAL PROTEIN QUALITY CONTROL 20 1.7 UBIQUITYLATION 25 2. RELEVANCE IN DISEASE 31 2.1 ERRORS IN GENE EXPRESSION CAN LEAD TO DISEASE 31 2.2 MISREGULATION OF UBIQUITYLATION RESULTS IN DISEASE 31 3. PROFILING THE BINDING SITES OF RNA-BINDING PROTEINS WITH NUCLEOTIDE RESOLUTION USING ICLIP 33 4. REFERENCES 56 II. AIMS OF THE PROJECT 62 III. INTERACTION PROFILING OF RNA-BINDING UBIQUITIN LIGASES REVEALS A LINK BETWEEN POSTTRANSCRIPTIONAL REGULATION AND THE UBIQUITIN SYSTEM 65 ABSTRACT 66 INTRODUCTION 66 RESULTS 68 STRATEGY FOR RECOVERING STABLE INTERACTION PARTNERS OF RNA-BINDING UBIQUITIN LIGASES 68 FUNCTIONAL COHERENCE OF THE IDENTIFIED INTERACTION PROFILE OF PRPF19 73 DEFINING THE INTERACTION PROFILES OF SELECTED RBULS 74 V Content DISCUSSION 81 MATERIAL AND METHODS 85 REFERENCES 91 SUPPLEMENTARY FIGURES 95 SUPPLEMENTARY TABLES 103 IV. THE RNA-BINDING UBIQUITIN LIGASE MKRN1 FUNCTIONS IN RIBOSOME-ASSOCIATED QUALITY CONTROL OF POLY(A)-TRANSLATION 111 ABSTRACT 112 INTRODUCTION 113 RESULTS 114 MKRN1 INTERACTS WITH PABPC1 AND OTHER RBPS 114 MKRN1 BINDS TO POLY(A) TAILS AND AT INTERNAL A-STRETCHES 117 MKRN1 PROMOTES RIBOSOME STALLING AT POLY(A) SEQUENCES 119 MKRN1 MEDIATES THE UBIQUITYLATION OF RIBOSOME-ASSOCIATED PROTEINS 120 DISCUSSION 123 PABP RECRUITS MKRN1 UPSTREAM OF A-STRETCHES AND POLY(A) TAILS 123 MKRN1 UBIQUITYLATES RPS10 AND TRANSLATIONAL REGULATORS TO STALL RIBOSOMES 123 DIFFERENCES BETWEEN HUMAN AND YEAST RQC EXPLAIN THE REQUIREMENT FOR MKRN1 125 MATERIALS AND METHODS 126 REFERENCES 138 SUPPLEMENTARY TABLES AND FIGURES 142 SUPPLEMENTARY TABLES 142 SUPPLEMENTARY FIGURES 144 V. FURTHER CHARACTERIZATION OF MKRN1 AND PRELIMINARY EXPERIMENTS 164 1. MATERIAL AND METHODS 165 1.1 MATERIALS 165 1.2 METHODS 177 2. RESULTS 185 2.1 MKRN1 CAN BE EFFICIENTLY DEPLETED IN HEK293T CELLS 185 2.2 MKRN1 PROTEINS ARE ECTOPICALLY OR STABLY EXPRESSED IN HEK293T CELLS 186 2.3 MKRN1 BINDS TO MRNA WITH MORE THAN ONE ZNF DOMAIN 187 2.4 CHANGES IN MKRN1 LEVELS REDOUND TO GLOBAL ALTERATIONS OF MRNA LEVELS 188 2.5 MKRN1 AFFECTS TRANSLATIONAL REGULATION 193 VI Content 2.6 MKRN1 UBIQUITYLATES PROTEINS FOUND WITHIN THE TRANSLATIONAL ELONGATING COMPLEX 198 2.7 MKRN1 LEVELS ARE REDUCED DURING STRESS CONDITIONS 202 3. REFERENCES 204 VI. DISCUSSION AND OUTLOOK 207 1. FUNCTIONAL INTERACTION NETWORK OF RBULS 207 2. MKRN1 IS INVOLVED IN DETECTING ABERRANT MRNAS, IN TRANSLATIONAL REGULATION AND HAS AN EFFECT ON MRNA STABILITY 208 2.1 MKRN1 IS A MRNP MEMBER 208 2.2 POLY(A) TAILS AND 3’ UTRS OF MRNAS ARE BOUND BY MKRN1 209 2.3 MKRN1 MEDIATES RIBOSOME STALLING 211 2.4 MKRN1 IS INVOLVED IN RQC AND UBIQUITYLATES RPS10 212 2.5 THE ROLE OF MKRN1 IN RQC MIGHT BE CONSERVED IN OTHER ORGANISMS 216 2.6 MKRN1 UBIQUITYLATES REGULATORS OF MRNA STABILITY AND TRANSLATION 218 2.7 MKRN1 MIGHT REGULATE TRANSLATION VIA THE 3’ UTR OR THE POLY(A) TAIL 220 2.8 MKRN1 LEVELS AFFECT MRNA ABUNDANCES 222 2.9 MKRN1 LEVELS ARE DOWNREGULATED IN RESPONSE TO STRESS 223 2.10 WHY MKRN1 IS IMPORTANT 224 2.11 SUMMARY 225 3. REFERENCES 226 VII. APPENDIX 230 ABBREVIATIONS 230 LIST OF FIGURES 234 LIST OF TABLES 235 VII Zusammenfassung ZUSAMMENFASSUNG Um ein funktionelles Protein herzustellen, wird DNA in Boten-RNA (mRNA) transkribiert. Diese wird dann posttranskriptionell verarbeitet und schließlich in ein Protein translatiert. Während der Genexpression wird die mRNA von RNA-bindenden Proteinen (RBPs), die das Schicksal der mRNA bestimmen, gebunden und verarbeitet. Eine besondere Klasse von RBPs sind RNA-bindende Ubiquitin-Ligasen (RBULs), welche Substratproteine ubiquitylieren können. Die zelluläre und molekulare Funktion der meisten RBULs ist kaum untersucht. Durch die Implementierung des adaptierten Affinitäts-Aufreinigung (AP)-Ansatzes werden in dieser Arbeit die Interaktions- Netzwerke von sechs RBULs bestimmt. Mit Hilfe der Genfunktions-Ähnlichkeits-Analyse werden die stabilen Bindungspartner von jedem der Köderproteine definiert. Die interagierenden Proteine geben Hinweise auf physiologische Funktionen dieser RBULs. Die identifizierten Interaktionspartner verbinden die Ubiquitylierungs-Maschinerie mit den verschiedenen Schritten des RNA-Metabolismus‘. RBULs verbinden also die molekularen Signalwege der post-transkriptionellen Regulation mit dem Ubiquitin- System. Das Ubiquitin-System ist am Abbau von defekten Proteinen beteiligt, um die Protein- Homöostase sicherzustellen. Schadhafte mRNAs, werden co-translationell erkannt. Im Rahmen dieser Arbeit habe ich herausgefunden, dass eine RBUL, das Makorin- Ringfinger-Protein 1 (MKRN1), an der Blockierung der Ribosomen an Poly(A)- Segementen während der Ribosom-assoziierten Qualitäts-Kontrolle (RQC) beteiligt ist. RQC ermöglicht den Abbau anomaler mRNAs und derer neu translatierten Polypeptide. Dies vermeidet die
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