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Untersuchung Zur Rolle Des La-Verwandten Proteins LARP4B Im Mrna- Metabolismus

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Untersuchung Zur Rolle Des La-Verwandten Proteins LARP4B Im Mrna- Metabolismus Untersuchung zur Rolle des La-verwandten Proteins LARP4B im mRNA-Metabolismus Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Maritta Küspert aus Schwäbisch Hall Würzburg 2014 Eingereicht bei der Fakultät für Chemie und Pharmazie am: ....................................... Gutachter der schriftlichen Arbeit: 1. Gutachter: Prof. Dr. Utz Fischer 2. Gutachter: Prof. Dr. Alexander Buchberger Prüfer des öffentlichen Promotionskolloquiums: 1. Prüfer: Prof. Dr. Utz Fischer 2. Prüfer: Prof. Dr. Alexander Buchberger 3. Prüfer: Prof. Dr. Stefan Gaubatz Datum des öffentlichen Promotionskolloquiums: ......................................................... Doktorurkunde ausgehändigt am: ................................................................................ Zusammenfassung Eukaryotische messenger-RNAs (mRNAs) müssen diverse Prozessierungsreaktionen durchlaufen, bevor sie der Translationsmaschinerie als Template für die Proteinbiosynthese dienen können. Diese Reaktionen beginnen bereits kotranskriptionell und schließen das Capping, das Spleißen und die Polyadenylierung ein. Erst nach dem die Prozessierung abschlossen ist, kann die reife mRNA ins Zytoplasma transportiert und translatiert werden. mRNAs interagieren in jeder Phase ihres Metabolismus mit verschiedenen trans-agierenden Faktoren und bilden mRNA-Ribonukleoproteinkomplexe (mRNPs) aus. Dieser „mRNP-Code“ bestimmt das Schicksal jeder mRNA und reguliert dadurch die Genexpression auf posttranskriptioneller Ebene. Für das La-verwandte Protein LARP4B (La-related protein 4B) wurde kürzlich eine direkte Interaktion mit den Translationsfaktoren PABPC1 (poly(A) binding protein, cytoplasmic 1) und RACK1 (receptor for activated C kinase) gefunden. Diese Befunde sowie die Assoziation mit aktiv translatierenden Ribosomen lässt vermuten, dass LARP4B zum mRNP-Code beiträgt. Die Domänenstruktur des Proteins legt darüber hinaus nahe, dass LARP4B direkt mRNAs bindet. Um einen Einblick in die Funktion von LARP4B und seiner in vivo RNA- Bindungspartner zu erhalten, wurde die mRNA-Assoziation transkriptomweit mit Hilfe von PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation)-Experimenten bestimmt. Diese Daten zeigten, dass LARP4B ein spezifisches Set an zellulären mRNAs über Sequenzbereiche in deren 3’- untranslatierten Regionen bindet. Die bioinformatische Auswertung der PAR-CLIP- Daten identifizierte ein LARP4B-Bindemotiv, welches durch in vitro Bindungsstudien validiert werden konnte. Darüber hinaus belegten pSILAC (pulsed stable isotope labeling with amino acids in cell culture)-Experimente und eine transkriptomweite Analyse der mRNA-Level, dass LARP4B die Expression der Ziel-mRNAs beeinflusst, indem es die Stabilität der gebundenen Transkripte erhöht. LARP4B konnte somit als positiver Faktor der eukaryotischen Genexpression identifiziert werden. Summary Eukaryotic messenger RNAs (mRNAs) undergo several processing reactions before they can serve as a template for protein biosynthesis. These processing reactions begin co-transcriptionally and include capping, splicing and polyadenylation. Once the processing is complete, the mature mRNA can be exported to the cytoplasm for translation. Throughout the various steps of the metabolism, the mRNAs interact with different sets of trans-acting factors to form mRNA ribonucleoprotein complexes (mRNPs). This “mRNP code” determines the fate of any mRNA and thereby regulates gene expression at the post-transcriptional level. Recently, the La-related protein 4B (LARP4B) was shown to interact directly with two translation factors, the cytoplasmic poly(A) binding protein 1 (PABPC1) and the receptor for activated C kinase (RACK1). These findings as well as its association with actively translating ribosomes suggest that LARP4B contributes to the mRNP code. The domain architecture of LARP4B suggests that LARP4B might bind mRNA directly. To gain insight into the function of LARP4B and its in vivo RNA binding partners, the associated mRNAs were determined using the transcriptome-wide PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) technology. These data show that LARP4B binds to a specific set of cellular mRNAs at the 3’-untranslated regions (3’-UTR). The biocomputational analysis of the PAR-CLIP data identified the LARP4B binding motif which could be validated by in vitro binding studies. Furthermore, taken together, pSILAC (pulsed stable isotope labeling with amino acids in cell culture) data and a transcriptome- wide analysis of mRNA levels demonstrate that LARP4B influences target mRNA expression by stabilizing the bound transcript. In conclusion, LARP4B was identified as a positive regulator for eukaryotic gene expression. Inhaltsverzeichnis 1 Einleitung ...................................................................................................................... 1 1.1 Messenger-RNA: Bindeglied zwischen der genetischen Information und der Proteinwelt .......................................................................................................................... 1 1.2 Der LeBenszyklus einer mRNA ..................................................................................... 2 1.3 Der AufBau des mRNP-Codes ........................................................................................ 4 1.4 Die Funktion der RBPs: Das Auslesen des mRNP-Codes ...................................... 6 1.5 mRNPs als Ursache für Krankheiten .......................................................................... 7 1.6 RNA-Bindedomänen vermitteln die Bindung von Proteinen an RNA ............. 8 1.7 Proteinfamilien RNA-bindender Proteine .............................................................. 10 1.8 Die Familie der La-verwandten Proteine ................................................................ 12 1.8.1 Die humanen LARPs .............................................................................................................. 14 1.1.1.1 Das genuine La-Protein ........................................................................................................... 14 1.1.1.2 Regulation der RNAP2-Transkription durch LARP7 .................................................... 14 1.1.1.3 LARP6 reguliert die Expression von Typ I Collagen ..................................................... 15 1.1.1.4 LARP1, ein Faktor der TOP-mRNA Expression ............................................................... 15 1.1.1.5 Die mRNP-Komponenten LARP4 und LARP4B ............................................................... 16 1.9 Problemstellung und Konzeption .............................................................................. 17 2 ErgeBnisse ................................................................................................................... 18 2.1 Bestimmung des LARP4B mRNA-Interaktoms mittels PAR-CLIP ................... 18 2.2 Validierung der PAR-CLIP-Daten ............................................................................... 22 2.3 LARP4B Bindet Bevorzugt 3’-untranslatierte Regionen .................................... 24 2.4 LARP4B interagiert mit AU-reichen Sequenzen ................................................... 25 2.5 LARP4B stabilisiert die gebundenen Ziel-mRNAs ................................................ 28 2.6 LARP4B stimuliert die Expession der Ziel-mRNAs .............................................. 30 2.7 LARP4B, LARP4 und LARP1 interagieren mit einem üBerlappenden Set an mRNAs ................................................................................................................................. 32 2.8 LARP4B, LARP4 und LARP1 agieren partiell redundant ................................... 33 3 Diskussion .................................................................................................................. 37 3.1 Identifizierung der in vivo Ziel-RNAs von LARP4B .............................................. 38 3.2 LARP4B interagiert Bevorzugt mit AU-reichen Sequenzen in der 3’-UTR ... 39 3.3 LARP4B stimuliert die Expression der Ziel-mRNAs durch StaBilisierung des gebundenen Transkripts .............................................................................................. 40 3.4 Der Beitrag von LARP4B zum mRNP-Code: eine Hypothese ............................ 42 3.5 LARP4B, LARP4 und LARP1: gleiche mRNA-Interaktoren, aBer unterschiedliche Funktionen? .................................................................................... 44 3.6 AusBlick .............................................................................................................................. 46 4 Material & Methoden .............................................................................................. 47 4.1 Materialien ........................................................................................................................ 47 4.1.1 Längenstandards .................................................................................................................... 47 4.1.2 Allgemeine Puffer ................................................................................................................... 47 4.1.3 Proteaseinhibitoren .............................................................................................................. 48 4.1.4 Medien ........................................................................................................................................
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