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Philipps-Universität Marburg
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Abstract
The eukaryotic cell nucleus posesses multiple morphologically and functionally different domains called nuclear compartments. Some of these nuclear compartments are e. g. the nucleolus and promyelocytic leukemia (PML) protein nuclear domains, which are dynamic subnuclear structures that play an important role in regulation of cell cycle, apoptosis, transcriptional regulation, tumor suppression and cellular antiviral response. The function as well as the regulation of processes emanating from these nuclear domains are not clearly understood so far. The aim of this dissertation was to identify and chracterize novel nuclear body associated proteins. These studies should allow a more detailed understanding of the function of nuclear domains.
In order to identify novel nuclear-dot localized proteins a straight foreward approach using the specific, dot-like nuclear localisation of so far not annotaded GFP-fused full length cDNAs has been chosen. Two of these proteins have been characerized as novel nuclear domain assoziated components.
One of the proteins, PAROT (PML-associated repressor of transcription)-protein, is a transcriptional repressor protein. It posesses a N-terminal repressor domain, a KRAB- (Krüppel associated box) domain, a linker-domain and a C-terminal C2H2-zincfinger (ZNF)-domain for DNA-binding, as well as three consensus-nuclear localization signals. Due to its domain structure PAROT belongs to the ZNF91-proteinfamily of "multiple-adjacent"-KRAB-C2H2-zinkfinger proteins, whereas the corresponding gene is lokalized on chromosome 19p13.11 as well as several other family members of the ZNF91-proteinfamily. PAROT mRNA was shown to be expressed as a 4.4 kb transcript in different human tissues like sceletal muscle, kidney, colon and brain. Ectopic expressed PAROT-protein localizes in nuclear domains and can be recruited through PML isoform PML IV in PML nuclear bodies. Furthermore it colocalizes with the corepressor TIF1b as well as members of heterochromatin protein 1 (HP1)-proteinfamily. PAROT seems to repress gene expression via a direct interaction with TIF1b as well as several other KRAB-ZNF repressor proteins. Recruitment of PAROT in PML bodies through PML IV leads to a dosisdependent modulation of PAROT-mediated transcriptional repression. Thus PAROT is a PML-body associated and due to that an adjustable transcriptional repressor.
anti-PAROT antibodies used in indirect immunofluorescent studies showed an unexpected mitochondrial localization of PAROT. This might be a hint that PAROT localizes to the nucleus only after a special stimulus to repress its target genes.
The second identified nuclear protein, Nucleostatmin, posesses two putative nuclear localization signals, a possible nucleolus localization sequence (NoLS) and a RNA-recognition motif (RRM). Thus Nucleostatmin is a RNA-binding protein. Nucleostatmin is highly conserved in mouse and rat and shows strong sequence homology in the RRM-containing part and in the C-terminal part harbouring the putative NoLS. Ectopic expressed Nucleostatmin localizes mostly to the nucleoplasm but also in part in nucleoli. The endogenous protein localizes together with RNA polymerase I and Upstream Binding Factor in fibrillar centres of nucleoli. Inhibition of DNA-dependent RNA sythesis by RNA polymerase I using Actinomycin D results in a diffuse distribution of Nucleostatmin in the nucleoplasma. Thus Nucleostatmin has a possible transcriptional or post-transcriptional function in rRNA transcription.
In summary two novel and unknown nuclear domain-associated proteins have been identified an characterizes with this experimental study. During the study it has been shown that localization of EYFP-fusionproteins not always corresponds to localization of endogenous proteins. Nevertheless for the PAROT protein as the first member of KRAB-C2H2 zincfinger proteins a PML-body association has been shown. Interestingly a second KRAB-C2H2, KRIM1A, is also recruited to PML bodies by PML IV. A PML-body association of Nucleostatmin still has to be investigated. This protein is probably a novel RNA-binding, nucleolar protein with a so far unknown function in rRNA transcription.
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Dates
Created: 2004Issued: 2004-08-12Updated: 2011-08-10
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
PML bodyKRAB-C2H2-ZinkfingerproteinNuclear domainNucleolus, RNA binding proteinPML-KerndomänenTranscription factor
DFG-subjects
RNS-BindungsproteineKernproteineNucleolusZellkernTranskriptionsfaktor
DDC-Numbers
570
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Fleischer, Sandra: Identifizierung neuer Kerndomänen assoziierter Proteine. : Philipps-Universität Marburg 2004-08-12. DOI: https://doi.org/10.17192/z2004.0438.
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This item has been published with the following license: In Copyright