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Sanjay Kumar Gupta The human CCHC-type Zinc Finger Nucleic Acid Binding Protein (CNBP) binds to the G-rich elements in target mRNA coding sequences and promotes translation Das humane CCHC-Typ-Zinkfinger-Nukleinsäure-Binde-Protein (CNBP) bindet an G-reiche Elemente in der kodierenden Sequenz seiner Ziel-mRNAs und fördert deren Translation Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences, Julius-Maximilians-Universität WürzBurg, Section: Biomedicine suBmitted By Sanjay Kumar Gupta from Varanasi, India WürzBurg, 2016 1 Submitted on: …………………………………………………………..…….. Office stamp Members of the Promotionskomitee: Chairperson: Prof. Dr. Alexander Buchberger Primary Supervisor: Dr. Stefan Juranek Supervisor (Second): Prof. Dr. Utz Fischer Supervisor (Third): Dr. Markus Landthaler Date of Public Defence: …………………………………………….………… Date of Receipt of Certificates: ………………………………………………. 2 Summary The genetic information encoded with in the genes are transcribed and translated to give rise to the functional proteins, which are building block of a cell. At first, it was thought that the regulation of gene expression particularly occurs at the level of transcription By various transcription factors. Recent discoveries have shown the vital role of gene regulation at the level of RNA also known as post-transcriptional gene regulation (PTGR). Apart from non-coding RNAs e.g. micro RNAs, various RNA Binding proteins (RBPs) play essential role in PTGR. RBPs have been implicated in different stages of mRNA life cycle ranging from splicing, processing, transport, localization and decay. In last 20 years studies have shown the presence of hundreds of RBPs across eukaryotic systems many of which are widely conserved. Given the rising numBer of RBPs and their link to human diseases it is quite evident that RBPs have major role in cellular processes and their regulation. The current study is aimed to descriBe the so far unknown molecular mechanism of CCHC-type Zinc Finger Nucleic Acid Binding Protein (CNBP/ZNF9) function in vivo. CNBP is ubiquitously expressed across various human tissues and is a highly conserved RBP in eukaryotes. It is required for emBryonic development in mammals and has Been implicated in transcriptional as well as post-transcriptional gene regulation; however, its molecular function and direct target genes remain elusive. Here, we use multiple systems-wide approaches to identify CNBP targets and document the consequences of CNBP Binding. We estaBlished CNBP as a cytoplasmic RNA-binding-protein and used Photoactivatable Ribonucleoside Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) to identify direct interactions of CNBP with 4178 mRNAs. CNBP preferentially Bound a G-rich motif in the target mRNA coding sequences. Functional analyses, including ribosome profiling, RNA sequencing, and luciferase assays revealed the CNBP mode of action on target transcripts. CNBP Binding was found to increase the translational efficiency of its target genes. We hypothesize that this is consistent with an RNA chaperone function of CNBP helping to resolve secondary structures, thus promoting translation. Altogether this study provides a novel mechanism of CNBP function in vivo and acts as a step-stone to study the individual CNBP targets that will Bring us closer to understand the disease onset. 3 Zusammenfassung Die in der DNA kodierte genetische Information wird transkriBiert und translatiert, um funktionelle Proteine zu Bilden, welche die Bausteine von Zellen sind. Lange Zeit wurde vermutet, dass die Regulation der Genexpression insBesondere auf dem Level der Transkription erfolgt. Kürzlich gemachte Entdeckungen haBen jedoch die zentrale Rolle der Genregulation auf dem Level der RNA, auch Bekannt als posttranskriptionelle Genregulation (PTGR), gezeigt. NeBen nicht-kodierenden RNAs wie microRNAs, besitzen verschiedene RNA-Binde-Proteine (RBP) eine Schlüsselrolle in der PTGR. RBPs wurden mit diversen EBenen des mRNA- LeBenszyklus, wie Speißen, Prozessieren, Transport, Lokalisation und ABBau in VerBindung geBracht. In den letzten 20 Jahren haben Studien die Existenz von Hunderten von RBPs in unterschiedlichen eukaryotischen Systemen gezeigt, von denen viele weithin konserviert sind. Bedenkt man die steigende Anzahl entdeckter und charakterisierter RBPs und ihren Bezug zu Krankheiten des Menschen, so ist es offensichtlich, dass RBPs eine große Rolle in der Regulation zellulärer Prozesse Besitzen. Das Ziel der hier vorliegenden Studie Bestand darin, die Bis jetzt unbekannten molekularen Mechanismen der Funktion des CCHC-Typ-Zinkfinger-Nukleinsäure- Binde-Proteins (CNBP/ZNF9) in vivo zu BeschreiBen. CNBP ist in verschiedenen humanen GeweBen uBiquitär exprimiert und ein hoch konserviertes RBP in Eukaryoten. Es ist für die emBryonale Entwicklung in Säugetieren notwendig und wurde mit der transkriptionellen und posttranskriptionellen Genregulation in VerBindung geBracht. Seine molekulare Funktion sowie die unmittelBaren Zielgene BlieBen jedoch unklar. In dieser Studie verwendeten wir systemweit analysierende Methoden um CNPB-Zieltranskripte zu identifizieren und dokumentierten die Folgen der Bindung von CNBP an diese. Wir haBen CNBP als ein zytoplasmatisches RNA-Binde-Protein charakterisiert und Quervernetzung und Immunpräzipitation mit photoaktivierBaren RiBonukleotiden (PAR-CLIP) angewendet. DaBei wurden direkte Interaktionen von CNBP mit 4178 mRNAs identifiziert. CNBP Bindet Bevorzugt an ein G-reiches Motiv in der kodierenden Sequenz der Ziel-mRNA. Funktionale Analysen, unter anderem RiBosom-Profil-Untersuchungen, RNA Sequenzierung und LuciferaseproBen, zeigten die Art und Weise, wie CNBP auf die Zieltranskripte wirkt. Die Bindung von CNBP an seine Zieltranskripte erhöht deren Translationseffizienz. Wir vermuten, dass dies eine RNA-Chaperon- Funktion von CNBP darstellt, die hilft Sekundärstrukturen aufzulösen und die Translation zu fördern. Zusammengefasst liefert diese Studie einen neuen Mechanismus der Funktion von CNBP in vivo und kann als Startpunkt dienen um einzelne CNBP Ziele zu untersuchen. Dies wird uns helfen dem Verständnis der Krankheitsentstehung näher zu kommen. 4 Table of contents 1 Introduction .............................................................................................................................................. 7 1.1 Regulation of gene expression in eukaryotes (overview) ......................................................................... 7 1.2 Post-transcriptional gene regulation (PTGR) .................................................................................................. 9 1.3 RNA Binding protein mediated regulation of gene expression ............................................................. 11 1.4 RNA Binding proteins in diseases ...................................................................................................................... 15 1.5 Zinc finger proteins .................................................................................................................................................. 17 1.6 CNBP as RNA Binding protein .............................................................................................................................. 18 1.7 Aim of the study ......................................................................................................................................................... 21 2 Results ...................................................................................................................................................... 23 2.1 HEK293 has simultaneously expressed isoforms of CNBP ..................................................................... 23 2.2 CNBP interacts with PRMT1 in vivo and is exclusively cytosolic ......................................................... 23 2.3 CNBP PAR-CLIP revealed its preference for CDS Binding ....................................................................... 25 2.4 CNBP interacts with mature mRNAs in G-rich regions ............................................................................. 27 2.5 CNBP Binds to G-rich sequences in vitro ......................................................................................................... 28 2.6 CNBP Binding slightly reduces target mRNA aBundance ........................................................................ 30 2.7 CNBP preferentially Binds to start of the target mRNAs .......................................................................... 32 2.8 RiBosome profiling shows stalling of translating riBosome on target mRNAs upon CNBP knockdown .......................................................................................................................................................................... 32 2.9 CNBP preferentially affects the translation of CDS targets without any site preference within CDS .......................................................................................................................................................................................... 34 2.10 CNBP knockdown reduces the target protein levels ............................................................................... 35 2.11 CNBP enhances the translation rate of target mRNAs By resolving the G-rich secondary structure ............................................................................................................................................................................... 36 3 Discussion ..................................................................................................................................................
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