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Regulation of gene silencing: From microRNA biogenesis to post-translational modifications of TNRC6 complexes DISSERTATION zur Erlangung des DOKTORGRADES DER NATURWISSENSCHAFTEN (Dr. rer. nat.) der Fakultät Biologie und Vorklinische Medizin der Universität Regensburg vorgelegt von Johannes Danner aus Eggenfelden im Jahr 2017 Das Promotionsgesuch wurde eingereicht am: 12.09.2017 Die Arbeit wurde angeleitet von: Prof. Dr. Gunter Meister Johannes Danner Summary ‘From microRNA biogenesis to post-translational modifications of TNRC6 complexes’ summarizes the two main projects, beginning with the influence of specific RNA binding proteins on miRNA biogenesis processes. The fate of the mature miRNA is determined by the incorporation into Argonaute proteins followed by a complex formation with TNRC6 proteins as core molecules of gene silencing complexes. miRNAs are transcribed as stem-loop structured primary transcripts (pri-miRNA) by Pol II. The further nuclear processing is carried out by the microprocessor complex containing the RNase III enzyme Drosha, which cleaves the pri-miRNA to precursor-miRNA (pre-miRNA). After Exportin-5 mediated transport of the pre-miRNA to the cytoplasm, the RNase III enzyme Dicer cleaves off the terminal loop resulting in a 21-24 nt long double-stranded RNA. One of the strands is incorporated in the RNA-induced silencing complex (RISC), where it directly interacts with a member of the Argonaute protein family. The miRNA guides the mature RISC complex to partially complementary target sites on mRNAs leading to gene silencing. During this process TNRC6 proteins interact with Argonaute and recruit additional factors to mediate translational repression and target mRNA destabilization through deadenylation and decapping leading to mRNA decay. Viral miRNA Biogenesis. Surprisingly, miRNAs were identified in human herpes, papilloma and polyoma viruses. These miRNAs regulate viral and host gene expression and influence infection efficiency. The miRNA biogenesis is strictly regulated and by northern blotting different expression profiles of infected cell lines were detected. To identify RNA-binding-proteins involved in post-transcriptional regulation of miRNA biogenesis, a mass spectrometric pull down assay with in-vitro transcribed pre-miRNA was established. The obtained data generated together with bioinformatical analyses a valuable set of potential regulatory candidates. The interaction of a subset of potential regulators was verified by repeating the pull-down with overexpressed Flag-/Ha-tagged proteins. For further functional characterization, the influence of RBPs on pre-miRNA processing was analyzed in knockout cell lines in which candidate RBPs have been depleted. Overexpression of the potential candidates further confirms a strong impact on the miRNA biogenesis. Taken together, mass spectrometric approaches identified RNA-binding-Proteins involved in viral miRNA biogenesis. Post-translational modifications of TNRC6 proteins. TNRC6 and Ago proteins play a central role in the gene silencing mechanism. The Interaction of both proteins is based on two Tryptophan’s binding into two specific pockets in the PIWI domain of Ago proteins. TNRC6 proteins (also referred to as GW proteins) contain Gly/Trp-repeats and serve as binding platform for many components of the gene silencing machinery. To assess whether gene silencing is regulated by post-translational modifications, TNRC6 proteins were analyzed by mass spectrometry. To analyze endogenous proteins, we established monoclonal antibodies against TNRC6A-C for immunopurificaiton of TNRC6 proteins from cell lysates. The validity and specificity of the antibodies was further verified by mass spectrometric selected reaction monitoring analyses. Followed by a detailed mass spectrometric analysis, multiple endogenous phosphorylation sites on TNRC6 proteins were detected. The obtained data identified conserved phosphorylation sites both among the TNRC6 paralogs and within different species. Functional analyses of phospho-mimicking and non-phospho mutants showed low effects on the downstream gene silencing processes. Localization studies and Ago-binding assays also indicate no effects of the phospho-sites on TNRC6 function. Taken together, post-translational modifications on TNRC6 proteins with potential, but so far unknown function in gene silencing were identified. Zusammmenfassung "From microRNA biogenesis to post-translational modifications of TNRC6 complexes" fasst die beiden Hauptprojekte dieser Doktorarbeit zusammen. miRNAs werden als primäre transkripte (pri-miRNA) von der RNA Polymerase II transkribiert. Die weitere Verarbeitung erfolgt durch den Mikroprozessor-Komplex, der das katalytisch aktive Enzym Drosha enthält, welches die pri-miRNA zu Vorläufer-miRNAs (pre-miRNA) spaltet. Nach dem Exportin-5-vermittelten Transport der pre-miRNA in das Zytoplasma, spaltet das RNase III- Enzym Dicer die terminale Schleife der pre-miRNA ab, was zu einer 21-24 nt langen doppelsträngigen RNA führt. Einer der beiden Stränge wird in den RNA-induzierten Silencing- Komplex (RISC) eingebaut, wo er direkt mit einem Mitglied der Argonaute-Proteinfamilie wechselwirkt. Der reife RISC-Komplex bildet durch komplementäre Basenpaarung der miRNA zur mRNA den Gene-silencing Komplexe. Während dieses Prozesses interagiert ein TNRC6-Protein mit Argonaut und durch Rekrutierung von zusätzlichen Faktoren wird die Translation reprimiert und die Ziel-mRNA destabilisiert und abgebaut. Virale miRNA Biogenese. Überraschenderweise wurden miRNAs bei humanen Herpes-, Papillom- und Polyomaviren identifiziert. Diese miRNAs regulieren die Virus- und Wirtsgenexpression und beeinflussen den viralen Lebenszyklus. Die miRNA-Biogenese ist streng reguliert und durch Nothern Blotting wurden verschiedene miRNA Expressionsprofile von infizierten Zelllinien nachgewiesen. Zur Identifizierung von RNA-bindenden Proteinen, die an der post-transkriptionelen Regulation der miRNA-Biogenese beteiligt sind, wurde eine massen-spektrometrische Pull-Down-Anwendung mit in vitro transkribierter Pre- miRNA etabliert. Die gewonnenen Daten, die zusammen mit bioinformatischen Analysen erzeugt wurden, sind ein wertvoller Datensatz von potenziellen regulatorischen Proteinen. Die Wechselwirkung einer Teilmenge von potentiellen Regulatoren wurde durch Wiederholen des Pull-downs mit überexprimierten Flag-/ Ha-markierten Proteinen verifiziert. Für eine weitere funktionelle Charakterisierung wurde der Einfluss von RNA-bindenden Proteinen (RBP) auf die pre-miRNA-Verarbeitung in Knockout-Zelllinien analysiert. Zusammenfasst, wurden in massenspektrometrischen Analysen RNA-bindende Proteine identifiziert, die an der viralen miRNA-Biogenese beteiligt waren. Posttranslationale Modifikationen von TNRC6-Proteinen. TNRC6- und Ago-Proteine spielen eine zentrale Rolle im Gen-Silencing-Mechanismus. Die Interaktion beider Proteine basiert auf zwei Tryptophan Bindungen, die in zwei spezifische Taschen in der PIWI-Domäne von Ago-Proteinen binden. TNRC6-Proteine (auch GW-Proteine genannt) enthalten repetitive Glycin/ Tryptophan- Aminosäureabschnitte und dienen als Bindeplattform für viele Komponenten der Gen-Silencing- Maschinerie. Um zu beurteilen, ob Gen-Silencing durch posttranslationale Modifikationen reguliert wird, wurden TNRC6-Proteine durch Massenspektrometrie analysiert. Um endogene Proteine zu analysieren, wurden monoklonale Antikörper gegen TNRC6A-C für Immuno-Aufreinigungen von TNRC6-Proteinen aus Zelllysaten etabliert. Die Gültigkeit und Spezifität der Antikörper wurde durch massenspektrometrische ausgewählte Analysen weiter verifiziert. Nach einer detaillierten Analyse wurden mehrere endogene Phosphorylierungsstellen in TNRC6-Proteinen nachgewiesen. Die erhaltenen Daten identifizierten konservierte Phosphorylierungsstellen sowohl unter den humanen TNRC6-Paralogen als auch innerhalb verschiedener TNRC6 proteine anderer Tiere. Funktionsanalysen von Phospho-Mimik- und Nicht-Phosphorylierbaren-Mutanten zeigten geringe Auswirkungen auf die nachgeschalteten Gen-Silencing-Prozesse. Lokalisierungsstudien und Ago- Bindungsversuche zeigen auch keine Wirkungen der phosphorylierten Aminosäuren auf die TNRC6-Funktion an. Zusammengefasst wurden posttranslationale Modifikationen an TNRC6-Proteinen identifiziert und charakterisiert. Publications Schraivogel D., Schindler S.G., Danner J., Kremmer E., Pfaff J., Hannus S., Depping R. & Meister G. Importin-β facilitates nuclear import of human GW proteins and balances cytoplasmic gene silencing protein levels. Nucleic Acids Res. 2015 Johannes Danner, Balagopal Pai, Ludwig Wankerl and Gunter Meister. Peptide-Based Inhibition of miRNA-Guided Gene Silencing. Methods Mol. Biol. 2017 Miguel Quévillon Huberdeau, Daniela M. Zeitler, Judith Hauptmann, Astrid Bruckmann, Lucile Fressigné, Johannes Danner, Sandra Piquet, Nicholas Strieder, Julia C. Engelmann, Guillaume Jannot, Rainer Deutzmann, Martin J. Simard and Gunter Meister. Phosphorylation of Argonaute proteins affects mRNA binding and is essential for microRNA-guided gene silencing in vivo. EMBO Journal 2017 Johannes Danner, Thomas Treiber, Nora Treiber, Emma Kraus, Eduard Hochmuth, Astrid Bruckmann, Christina Paulus, Michael Nevels, Hans-Helmut Niller, Adam Grundhoff and Gunter Meister. Seduction of viral miRNA biogenesis during viral life cycle Manuscript in preparation Mariangela Morlando, Sama Shamloo, Johannes Danner, Astrid Bruckmann, Gunter Meister and Irene Bozzoni. The Interplay between lnc31, pre-lnc31 and YBox1 in differentiating muscle cells
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