The PHD finger protein 5 is a part of the spliceosome and acts as a DNA-binding protein
Proteininteraktionen, Analyse der Expression und Funktionsanalyse der PHF5a-Protein
by Tomasz Rzymski
Date of Examination:2004-11-03
Date of issue:2005-12-06
Advisor:Prof. Dr. Dr. Wolfgang Engel
Referee:Prof. Dr. Wolfgang Engel
Referee:Prof. Dr. Christiane Gatz
Referee:Prof. Dr. Wolfgang Liebl
Persistent Address:
http://dx.doi.org/10.53846/goediss-616
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Abstract
English
Four proteins namely U2AF35, SRp40, mDomino and Ddx1 were identified as PHF5a interacting partners by using the murine 11.5-days embryo yeast two-hybrid library screening.PHF5a interacting domains of SR proteins U2AF35 and SRp40 were restricted to Cterminal arginine-serine rich domains (RS).Other members of the SR protein family namely SRp20, SRp30c and ASF/SF2 were shown that they can not bind to the PHF5a protein by using a directed yeast twohybrid assay and the a-galactosidase assay.Protein interactions between PHF5a and U2AF35, SRp40, mDomino and Ddx1 were verified by using in vitro coimmmunoprecipitation assays.Mapping of binding sites by using coimmunoprecipitation experiments and a directed yeast two-hybrid assay demonstrated that both ATP-dependent helicases mDomino and Ddx1 interact with the C-terminal segment of PHF5a. In addition, the N-terminal part of PHF5a was identified as a region responsible for binding to splicing proteins U2AF35 and SRp40. By using the yeast-three hybrid assay it was demonstrated that PHF5a is able to play a role as a bridge protein which can bind splicing proteins U2AF35, SRp40 and ATPdependent helicases Ddx1, mDomino simultaneously.immunostaining on NIH3T3 cells demonstrated that the PHF5a protein is localized predominantly in the nucleus of stained cells.Signals from both the PHF5a-GFP fusion protein and for U2AF35 or SRp 40 partially colocalize and are distributed in multiple speckles throughout the nucleus.Four splicing forms of U2AF35 were isolated by RT-PCR and the yeast two-hybrid experiment and characterized.Expression of PHF5a and U2AF35 proteins in the testis were analyzed. It was demonstrated that PHF5a shows a time and spatial coexpression with the U2AF35 protein predominantly in the nuclei of pachytene spermatocytes. In addition, the PHF5a protein was found to be up-regulated in spermatocyte-specific GC-4spc cells as compared to spermatogonia- specific GC-1spg cells.PHF5a and U2AF35 interact in vivo in spermatocyte-derived GC-4spc cells which was demonstrated by using in vivo coimmunoprecipitation assays.By using the in vivo splicing assay we could demonstrate that the PHF5a protein does not directly affect splicing of the ß-globin mini gene.Whole mount in situ hybridization on 11.5-days mouse embryos suggests that PHF5a is expressed ubiquitously during embryonic development.A PHF5a-GST fusion protein was able to pull down genomic DNA. Furthermore, by using the whole genome PCR and SELEX putative target DNA sequences of the PHF5a protein were identified. Down-regulation of PHF5a expression in NIH3T3 cells by using the RNAi technique inhibit cell proliferation.
Keywords: PHF5a; U2AF35; splicing; spliceosom; DNA-binding
Other Languages
Vier Proteine: U2AF35, SRp40, mDomino und Ddx1 wurden durch Screening Yeast-two hybride-Bibliothek als Partner des PHF 5a-Protein gefunden. Proteininteraktionen wurden in-vitro und in-vivo bestätigt. Die für Bindungen verantwortlichen Domäne wurden als RS Domäne (reich an Arginin-Serin) identifiziert. PHF5a funktioniert als "Brücke" zwischen ATP-Helikasen mDomino und Ddxl und Splicing Proteinen U2AF35 und SRp40. Forschungen Experimente mit der Anwendung von Fusions-Proteine und der Methode Färbung mit Antikörper haben bewiesen, dass PHF5a ein Zellkernprotein ist und mit U2AF35 und SRp40 kolokalisiert. Es wurde zusätzlich das Profil von Expression der Proteinen PHF5a und U2AF35 während der Embryoentwicklung und der Postnatalzellkernentwicklung analisiert
Schlagwörter: Proteininteraktionen; Analyse der Expression und Funktionsanalyse der PHF5a-Protein