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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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Defects in DNA repair and RNA metabolism associated with human neurological disorders
Cihlářová, Zuzana ; Hanzlíková, Hana (advisor) ; Čermák, Lukáš (referee) ; Roithová, Adriana (referee)
The human genome is constantly under the attack by various damaging agents, leading to the breakage of one or both strands of DNA that might interfere with RNA processing. Importantly, our cells have evolved diverse mechanisms to rapidly repair various DNA lesions, highlighting the importance of genetic integrity. Defects in DNA repair and/or RNA metabolism can lead to a variety of human hereditary diseases, with pathologies including growth and developmental defects, immunodeficiency, predisposition to cancer, and neurodegeneration. Mutations in the BRAT1 (BRCA1-associated ATM activator-1) protein have been associated with neurological disorders characterized by heterogenous phenotypes with varying levels of clinical severity ranging from microcephaly, hypertonia, epilepsy, seizures, and early death in the first two years of life to mild cerebellar atrophy and ataxia. Previously, BRAT1 protein has been implicated in the cellular response to DNA double-strand breaks and ATM signalling. However, the exact mechanism/s by which mutations in BRAT1 gene trigger neurological disorders are largely unknown. Recently, we have identified a homozygous missense c.185T>A (p.Val62Glu) variant in BRAT1 that markedly reduced the level of BRAT1 protein in patient-derived cell lines. Surprisingly, our data show that...
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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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