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Role of PML in ribosomal stress
Kremserová, Petra ; Vašicová, Pavla (advisor) ; Sztacho, Martin (referee)
PML is involved in many cellular processes. It organizes nuclear structures PML nuclear bodies (PML NBs) and it associates with nucleolus in response to ribosomal stress to form PML nucleolar associations (PNAs). The function of PNAs is unclear. To elucidate this question, one can attempt to identify proteins interacting with PML at nucleolus. The common method is co- immunoprecipitation, however, this approach cannot be used for PML due to its low solubility. To defeat this, an alternative way of proximity-dependent biotin labelling could be used. The goal of this work was to explore a suitability of biotin labelling for identification of PML nucleolar partners. For this purpose I prepared constructs of wild type or mutated PML with GFP and biotin ligase for transient and stable expression and analysed their propensity to form PML NBs and doxorubicin-induced PNAs, and biotinylate their vicinity. In transient expression, both fusion proteins formed PML NBs and only wild type but not mutated PML IV formed PNAs after doxorubicin treatment with preserved biotinylation capability. In stable expression of fusion proteins in cells with PML knockout the number and composition of PML NBs was aberrant and no PNAs were observed. However, this system was utilized for optimization of solubilisation of biotinylated...
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The role of evolutionarily conserved proteins BIR-1/Survivin and SKP-1 in the regulation of gene expression
Kostrouch, David ; Kostrouch, Zdeněk (advisor) ; Dráber, Pavel (referee) ; Pacák, Karel (referee)
SKIP and BIR/Survivin are evolutionarily conserved proteins. SKIP is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Loss of function of C. elegans SKIP (SKP-1) and BIR-1 induces overlapping developmental phenotypes. In order to uncover the possible interactions of SKP-1 and BIR-1 on the protein level, we screened the complete C. elegans mRNA library using the yeast two-hybrid system. These experiments identified partially overlapping categories of proteins as SKP-1 and BIR-1 interactors. The interacting proteins included ribosomal proteins, transcription factors, translation factors and cytoskeletal and motor proteins suggesting involvement of the two studied proteins in multiple protein complexes. To visualize the effect of BIR-1 on the proteome of C. elegans we induced a short time pulse BIR-1 overexpression in synchronized L1 larvae. This led to a dramatic alteration of the whole proteome pattern indicating that BIR-1 alone has the capacity to alter the chromatographic profile of many target proteins including proteins found to be interactors in yeast two hybrid screens. The results were validated for ribosomal proteins RPS-3, RPL-5, non-muscle myosin and TAC-1, a transcription cofactor and a centrosome associated...
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Role of PML in ribosomal stress
Kremserová, Petra ; Vašicová, Pavla (advisor) ; Sztacho, Martin (referee)
PML is involved in many cellular processes. It organizes nuclear structures PML nuclear bodies (PML NBs) and it associates with nucleolus in response to ribosomal stress to form PML nucleolar associations (PNAs). The function of PNAs is unclear. To elucidate this question, one can attempt to identify proteins interacting with PML at nucleolus. The common method is co- immunoprecipitation, however, this approach cannot be used for PML due to its low solubility. To defeat this, an alternative way of proximity-dependent biotin labelling could be used. The goal of this work was to explore a suitability of biotin labelling for identification of PML nucleolar partners. For this purpose I prepared constructs of wild type or mutated PML with GFP and biotin ligase for transient and stable expression and analysed their propensity to form PML NBs and doxorubicin-induced PNAs, and biotinylate their vicinity. In transient expression, both fusion proteins formed PML NBs and only wild type but not mutated PML IV formed PNAs after doxorubicin treatment with preserved biotinylation capability. In stable expression of fusion proteins in cells with PML knockout the number and composition of PML NBs was aberrant and no PNAs were observed. However, this system was utilized for optimization of solubilisation of biotinylated...
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The role of evolutionarily conserved proteins BIR-1/Survivin and SKP-1 in the regulation of gene expression
Kostrouch, David ; Kostrouch, Zdeněk (advisor) ; Dráber, Pavel (referee) ; Pacák, Karel (referee)
SKIP and BIR/Survivin are evolutionarily conserved proteins. SKIP is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Loss of function of C. elegans SKIP (SKP-1) and BIR-1 induces overlapping developmental phenotypes. In order to uncover the possible interactions of SKP-1 and BIR-1 on the protein level, we screened the complete C. elegans mRNA library using the yeast two-hybrid system. These experiments identified partially overlapping categories of proteins as SKP-1 and BIR-1 interactors. The interacting proteins included ribosomal proteins, transcription factors, translation factors and cytoskeletal and motor proteins suggesting involvement of the two studied proteins in multiple protein complexes. To visualize the effect of BIR-1 on the proteome of C. elegans we induced a short time pulse BIR-1 overexpression in synchronized L1 larvae. This led to a dramatic alteration of the whole proteome pattern indicating that BIR-1 alone has the capacity to alter the chromatographic profile of many target proteins including proteins found to be interactors in yeast two hybrid screens. The results were validated for ribosomal proteins RPS-3, RPL-5, non-muscle myosin and TAC-1, a transcription cofactor and a centrosome associated...
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