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CSL proteins of Schizosaccharomyces pombe
Převorovský, Martin ; Půta, František (advisor) ; Dvořák, Michal (referee) ; Kořínek, Vladimír (referee)
The CSL family of transcription factors is essential for metazoan development,mostly due to their involvement in the Notch signaling pathway. We identified two novel classes of CSL genes in several fungal species, organisms lacking the Notch pathway. We characterized experimentally cbfl I- and cbfl2*, the two CSL genesof Schizosaccharomyces pombe, in order to elucidate the CSL function in fungi. We provide evidence supportingtheir identiý as genuine CSL genes.Both cbfll- and cbfl2- are non-essential; they have distinct expression profiles and code for nuclear proteins with transcription activation potential. Significantly, we demonstratedthat Cbfl1 recognizesspecifically the canonical CSL response element GTGA/6GAA in vitro. The deletion of cbfLt_ is associated with growth phenoýpes and altered colony morphology. Furthermore, we found that Cbfl I and CbfIZ play opposite roles in cell adhesion, nuclear and cell division and their coordination. Disturbed balance of the two CSL proteins leads to cell separationdefects,cut phenotype, and high-frequency diploidization. Our data show that CSL proteins operate in an organism predating the Notch pathway, which should be of relevance to the understandingof (Notch- independent)CSL functions in metazoans.
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Regulation of S. cerevisiae TUB1 and TUB3 paralogous genes expression by Prp45
Cihlářová, Zuzana ; Půta, František (advisor) ; Schierová, Michaela (referee)
Prp45 is an essential splicing factor of budding yeast Saccharomyces cerevisiae. The human ortholog of Prp45 - protein SNW1/SKIP - is involved in splicing and probably influences transcription and histone modification. The genetic interacion of Prp45 with splicing factors is well described. We have additionally demonstrated that Prp45 genetically interacts also with factors involved in transcription regulation and histone modification enzymes. Our preliminary data therefore suggest that Prp45 might be a factor that connects processes of splicing, transcription, and chromatine modification and dynamics in S. cerevisiae. The first aim of this project was to investigate the role of introns in intra- and intergenic expression regulation of paralogous genes TUB1 and TUB3 and whether is this regulation influenced by aberrant splicing. Using quantitative PCR we found that expression of paralogous genes TUB1 and TUB3 is not dependent on the presence of their introns or correct splicing. The second aim of this project was to explore the potential role of Prp45 in the regulation of chromatin state. For this purpose, we used the system of β-estradiol-induced expression of myc-tagged histon H3 and determined its incorporation into nucleosomes by chromatin immunoprecipitation. Despite the lack of...
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Does the pre-mRNA splicing occur in S. cerevisiae co- or post-transcriptionally?
Cihlářová, Zuzana ; Půta, František (advisor) ; Kozáková, Eva (referee)
Until recently, the splicing and transcription were seen as almost independent processes. However, today a lot of studies provide plenty of evidence about their connection, even in the yeast Saccharomyces cerevisiae. The connection of these processes is particularly mediated by C-terminal domain of RNA polymerase II, which is consisted of tandemly repeated heptapeptide sequence - YSPTSPS. Amino acid residues of this heptapeptide sequence are specifically phosphorylated during transcription, which regulates transcription process and also the binding of specific factors. These factors are necessary for processing of the nascent transcript. Modifications of the primary transcript occur especially cotranscriptionally in higher eukaryotes, thus before the transcription is terminated and also before the functional mRNA is released. Opinion on cotranscriptional splicing in S. cerevisiae were significantly changed in the last years. However, nowadays the splicing of pre-mRNA of most genes in S. cerevisiae is seen as cotranscriptional process. RNA polymerase II pauses within the terminal exons and this pausing event provides sufficient time for each spliceosomal component to assemble on the pre-mRNA and also for catalysis of splicing before the transcription termination. Keywords: cotranscriptional...
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Role of promoter in the regulation of alternative splicing
Kozáková, Eva ; Staněk, David (advisor) ; Půta, František (referee) ; Blažek, Dalibor (referee)
It was shown that 95 % of human multi-exon genes are alternatively spliced and the regulation of alternative splicing is extremely complex. Most pre-mRNA splicing events occur co- transcriptionally and there is increasing body of evidence, that chromatin modifications play an important role in the regulation of alternative splicing. Here we showed that inhibition of histone deacetylases (HDACs) modulates alternative splicing of ~700 genes via induction of histone H4 acetylation and increase of Pol II elongation rate along alternative region. We identified HDAC1 the catalytic activity of which is responsible for changes in alternative splicing. Then, we analyzed whether acetylhistone binding protein Brd2 regulates alternative splicing and showed that Brd2 occupies promoter regions of targeted genes and controls alternative splicing of ~300 genes. Later we showed that knockdown of histone acetyltransferase p300 promotes inclusion of the alternative fibronectin (FN1) EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as the p300 knockdown. Next we showed that p300 controls histone...
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GAL4/UAS binary system as a toll for the study of gene function
Soukup, Tomáš ; Krylov, Vladimír (advisor) ; Půta, František (referee)
GAL4/UAS system is a bipartite gene engineering tool, enabling ectopic expression in temporal and tissue-specific manner in vivo. Design of this technique is based on a mechanism of gene transcription, which was elucidated of large portion by an experimental study of Saccharomyces cerevisiae regulation of metabolic control circuit for processing galactose. It is possible to generate hundreds of stable transgenic lines by independent incorporation of the gene for the transcription factor Gal4p and its binding sequence (UAS), respectively, by using transposible or specific-sequence integration techniques. An individual organism, manifesting ectopic expression in suitable, adjustable conditions, can be obtained by cross breeding individual of GAL4 lines with individual from UAS line. This thesis represents a synthesis of the basic principles of GAL4/UAS system and its variants, particularly adapted to the needs of genetic manipulation of model organisms Drosophila melanogaster and Danio rerio. Additionally, this text summarizes the connection GAL4/UAS system with other techniques and briefly highlights the potential for practical applications mainly in research area of neurology. Powered by TCPDF (www.tcpdf.org)
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Localization matters: function of paxillin and phopholipids in the cell nucleus
Marášek, Pavel ; Hozák, Pavel (advisor) ; Půta, František (referee) ; Žárský, Viktor (referee)
(English) Both paxillin and PIP2 are well known components of the cell, although of a distinct origin. Focal adhesion protein paxillin spreads the signals from extracellular matrix via integrins and growth factor receptors to affect cellular motility and migration (Schaller, 2001). PIP2, a major structural component of cytoplasmic membrane, is utilized by phospholipase C to generate second messenger molecules (Hokin and Hokin 1953; Streb et al. 1983). Both molecules were recently shown to be localized in the nucleus. Their original functions have been well established, but together with other research colleagues we are now shedding more light on completely different functions of these biological molecules and moreover, in the different compartments than they were primarily believed to function in. Here, we introduce paxillin as an important factor of the cell nucleus, where it regulates transcription of two important growth-related genes, IGF2 and H19. It does not affect the allelic expression of these imprinted genes, it rather regulates long-range chromosomal interactions between H19 or IGF2 promoter, and the shared distal enhacer on an active allele. In detail, paxillin stimulates the interaction between the enhancer and the IGF2 promoter, activating IGF2 gene transcription, while it restrains...
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The influence of intron sequences on splicing effectivity in Saccharomyces cerevisiae
Oplová, Michaela ; Půta, František (advisor) ; Malcová, Ivana (referee)
Pre-mRNA splicing is a highly regulated cellular process. The tight cooperation of spliceosome and other splicing factors that enable pre-mRNA cis-elements interpretation results in precise pre-mRNA splicing regulation. Short conserved splicing sequences within introns represent an elementary and indispensable element for intron removal from primary transcript, yet they are not sufficient signals for efficient splicing events. Additional pre-mRNA features affect complex splicing regulation. We took advantage of strains with slightly disrupted spliceosome (prp45(1-169)) to study the effect of ACT1 and MAF1 intronic sequences on splicing efficiency. Here we show, that ACT1 intron region between branch point (BP) and 3' splice site (3'ss) maintains splicing efficiency in mutant cells. However, the specific element within this region was not determined. In addition, results implicate an alternative BP in splicing efficiency modulation in yeast Saccharomyces cerevisiae. Interestingly, this alternative BP is localized in ACT1 intron outside of the BP-3'ss region. Furthermore, splicing factors with potential influence on 3'ss selection were studied. Heterodimer composed of Slu7p and Prp18p participates in 3'ss positioning to the active site of the spliceosome. Splicing analysis of substrates with two...
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Aptamers - binding and regulation abilities of RNA molecules
Oplová, Michaela ; Půta, František (advisor) ; Dzijak, Rastislav (referee)
Aptamers are single stranded ribo- or deoxyribonucleotides usually 20 to 80 nucleotides in length that occupy a complex three-dimensional structures by intramolecular interactions and bind to small target molecules with high affinity and specificity. Aptamers are generated in vitro using revolutionary technology SELEX (systematic evolution of ligand by exponential enrichment) and its modifications. They have recently attracted considerable attention of the scientific and medical community because of the fact that is possible to prepare aptamers binding practically any target molecule and this is making aptamers promising as diagnostic and therapeutic tools. Aptamers also exist naturally; aptamer domains have been found repeatedly as part of the regulatory elements of gene expression in bacteria, where they act as specific receptors for cellular metabolites. Domain TPP has been also found in plants, fungi and green algae.
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Preparation and testing of polyclonal antibodies against Cbf11 and Cbf12, the fission yeast CSL transcription factors
Tvarůžková, Jarmila ; Půta, František (advisor) ; Moserová, Michaela (referee)
CSL (CBF1/RBP-Jκ, Suppressor of Hairless, Lag-1) protein family members are transcription factors critical for metazoan development as the effectors of Notch signaling pathway as well as in a Notch-independent manner. CSL homologues have been identified in fungal organisms lacking the Notch signaling pathway. Cbf11 and Cbf12 are antagonistic paralogous proteins that are important for proper coordination of cell and nuclear division, regulation of cell adhesion and chromosome integrity in the fission yeast Schizosaccharomyces pombe. The activities of Cbf11 and Cbf12 proteins need to be finely balanced for their proper biological function, however, chromosomally tagged alleles of these proteins exhibit properties different from wild type. Therefore, the availability of specific antibodies would greatly enhance the study of CSL proteins in the fission yeast. In this bachelor's thesis, design and preparation of imunogen for commercial antibody production followed by antibody testing is presented. Using bioinformatics, suitable immunogenic Cbf11 and Cbf12 protein fragments were selected and the corresponding DNA sequences were cloned into an expression vector. His-tagged expression was optimized in a bacterial expression system and the native protein was purified using immobilized metal affinity...
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Regulation of pre-mRNA splicing in S. cerevisiae: where RNA cooperates with proteins.
Gahura, Ondřej ; Půta, František (advisor) ; Pospíšek, Martin (referee) ; Staněk, David (referee)
Ondřej Gahura, PhD Thesis 2011 Regulation of pre-mRNA splicing in S. cerevisiae: where RNA cooperates with proteins Abstract Removal of introns from protein coding transcripts occurs in two splicing reactions catalyzed by a large nuclear complex, spliceosome. The spliceosome is an extremely intricate and dynamic machine, wherein contributions of small RNA molecules and multiple proteins are coordinated to meet the requirements of absolute precision and high flexibility. For an intimate understanding of pre-mRNA splicing, it is necessary to unravel roles of individual components and to dissect the partial mechanisms. In the first part of this work, we describe the role of the Prp45 splicing factor in Saccharomyces cerevisiae. Mapping of genetic interactions of a conditionally lethal allele prp45(1-169) suggests a relationship of Prp45 to the NTC complex and to the second transesterification. Two-hybrid assay and purification of spliceosomal complexes reveal a contribution of the Prp45 C-terminus in the Prp22 helicase recruitment and/or regulation. Numerous experiments with reporter substrates document the need of Prp45 for the efficient splicing of a specific subset of introns. Our observations suggest that the function of Prp45 in splicing is conserved in evolution. The second part is devoted to the role of...
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