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Role of the WIP1 phosphatase in the nucleolus
Palková, Natálie ; Macůrek, Libor (advisor) ; Sztacho, Martin (referee)
Protein phosphatase 2C delta (known as WIP1) is an important negative regulator of the DNA damage response (DDR) signalling. As a chromatin-bound protein, it dephosphorylates and thus inactivates ATM kinase and the transcription factor p53. Increased expression of WIP1 suppresses the function of the tumour suppressor p53 and contributes to the development of several cancer types, including breast and brain tumours. In recent years, it has been shown that WIP1 can also regulate cellular processes that are not directly linked to DDR, such as ensuring telomere stability. However, alternative functions of the WIP1 protein have not yet been fully understood. In this work, we described a novel role of the WIP1 phosphatase in the nucleolus, the organelle responsible for ribosome biogenesis. We found that WIP1 associates with many nuclear proteins, and using deletion mutants, we identified a nucleolar localisation sequence (NoLS) at the C-terminus of the protein. Using super-resolution microscopy, we detected the localisation of WIP1 phosphatase in the fibrillar centres of the nucleolus. We employed an inducible Cas9 system for generating double-strand breaks in ribosomal DNA and found that WIP1 has an impact on recruitment of DNA repair factors to the nucleolar caps. Analysis based on quantitative...
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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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lncRNA MIAT expression in cancer cells.
Jursová, Pavlína ; Eckschlager, Tomáš (advisor) ; Sztacho, Martin (referee)
LncRNAs have been shown, in many cases, to function as important regulators for gene expression and thus, they can play a critical role in various biological functions and disease processes including cancer. Myocardial infarction associated transcript (MIAT) is one of the non-coding RNAs first identified as lncRNA in 2006 and originally isolated as a candidate gene for myocardial infarction. This long non-coding RNA is also involved in other diseases such as diabetic retinopathy, paranoid schizophrenia or microvascular dysfunction. MIAT has also been identified as a carcinogenic regulator in many malignant tumors. Numerous researches have reported that MIAT silencing reduces cell viability, proliferation and invasivity and enhances cellular senescence and apoptosis of cancer cells. Therefore, it is considered a potential biomarker and therapeutic target in cancer. MIAT is involved in cellular processes through various mechanisms. It regulates alternative splicing, gene expression or functions through ceRNA mechanism and thus influences biological processes related to the tumor formation. Furthermore, in this study have been found that relative expression of MIAT was increased in Ewing sarcoma cell lines.
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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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