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Chromatin modifiers and their relation to transcription regulation in Saccharomyces cerevisiae
Hálová, Martina ; Folk, Petr (advisor) ; Staněk, David (referee)
Relations among transcription, pre-mRNA processing and chromatin modifications are only partially understood. The human protein SNW1/SKIP belongs to factors which couple these processes. The protein plays role in pre-mRNA splicing and transcription on the level of both initiation and elongation. According to the hypothesis of K. Jones laboratory, it physically and functionally interacts with positive transcription elongation factor b during transcription elongation and influences methylation of histone H3 on lysine 4, a modification characteristic for active transcription (Bres et al., Genes Dev. 19:1211-26, 2005, Bres et al., Mol Cell. 36:75-87, 2009). The yeast ortholog of SNW1/SKIP, Prp45, was until now reported only in connection with splicing regulation. However, unpublished results from our Laboratory and others showed that it is employed in transcription elongation as well. The aim of the diploma project was to search for the relations between Prp45 and the factors regulating transcription. It was confirmed that the mutation prp45(1 169) results in the delay of PHO5 and PHO84 expression during transcriptional induction. Next, we discovered new genetic interactions between PRP45 and several genes encoding the effectors of chromatin modifications. How Prp45 influences the expression of PHO5 and PHO84...
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Analýza regulace komplexů cytoplazmatických poly(A) polymeráz
Novák, Jakub ; Staněk, David (advisor) ; Hrossová, Dominika (referee)
The regulation of gene expression is achieved at many levels. Chromatin-based gene regulation has been the central focus of many decades of research; however, posttranscriptional control mechanisms are emerging as a fundamental complement to direct protein synthesis. This thesis is focused on a specific mechanism of posttranscriptional control - the translational regulation of mRNAs in the cell cytoplasm. This control is a consequence of the balance between translational repression and activation and hinges on the selective recognition of regulated mRNAs by RNA-binding proteins and their ability to recruit RNA modifying proteins. In this thesis, Caenorhabditis elegans germline was used to study translational control of the germ cell-enriched gene, gld-2. Mutants of known RNA-binding proteins of the PUF and CPB protein families were analyzed by performing Western blots, using anti-GLD-2 antibodies. Yeast 3-Hybrid system was used to identify the cis-regulatory sites in the gld-2 mRNA conferring translational regulation by members of PUF and CPB protein families. Potential autoregulatory loop of gld-2 gene expression was also investigated. This thesis shows that FBF proteins positively regulate expression of gld-2 and bind to a conserved sequence in the 3'UTR of its mRNA. Mutations of gld-2 negatively affect...
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Spliceosome assembly
Hausnerová, Viola ; Staněk, David (advisor) ; Chalupníková, Kateřina (referee)
Pre-mRNA splicing is a process in which introns are removed from eukaryotic transcripts and exons are ligated together. Splicing is catalyzed by spliceosome, a large ribonucleoprotein complex composed of five small nuclear RNAs and more than 100 additional proteins, which recognizes 5' splice site, branch point site and 3' splice site and performs two transesterification reactions to produce mRNA molecules. 5' splice site is recognized by U1 snRNP and U2 auxiliary factor (U2AF) is involved in branch point and 3' splice site recognition in the early splicing complex. There is some evidence of splice sites cooperation during intron recognition in vitro but little is known about the situation in vivo. Using Fluorescence resonance energy transfer (FRET) and RNA immunoprecipitation (RIP) methods, we have investigated the early stages of spliceosome assembly. We have employed splicing reporters based on -globin gene and MS2 stem loops to detect interactions of proteins on RNA molecule directly in the cell nucleus. Results of FRET indicate that intact 5' splice site is required for U2AF35 interaction with 3' splice site and that U1C recruitment to 5' splice site is partially limited upon 3' splice site mutation. We have also confirmed by RIP that U2 snRNP association with pre-mRNA molecule requires presence of 5'...
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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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Regulace pre-mRNA sestřihu v prostředí buněčného jádra
Hnilicová, Jarmila ; Staněk, David (advisor) ; Půta, František (referee) ; Dvořák, Michal (referee)
Eukaryotic genes contain non-coding sequences - introns that are removed during pre-mRNA splicing by the spliceosome. The spliceosome is composed of five snRNPs (U1, U2, U4/U6 and U5) which assemble on pre-mRNA in a step-wise manner and together with additional non-snRNP proteins catalyse splicing. Mutations in splicing factors can cause severe diseases, for example a point missense mutation (called AD29) in hPrp31 (U4/U6 snRNP specific protein) induces retinitis pigmentosa, disease often leading to complete blindness. In this PhD thesis we show that the hPrp31 AD29 mutant is unstable and is not properly incorporated into spliceosomal snRNPs. In addition, the expression of the mutant protein reduces cell proliferation, which indicates that it interferes with cellular metabolism (likely splicing) and could explain the induction of retinitis pigmentosa. Next, we focus on a role of nuclear environment in pre-mRNA splicing. It was shown that new U4/U6·U5 snRNPs are preferentially assembled in non-membrane nuclear structure - Cajal body. Here we expand this finding and provide evidence that Cajal bodies are also important for U4/U6·U5 snRNP recycling after splicing. In addition, we analyzed a role of chromatin and particularly histone acetylation modulates in splicing regulation. Using inhibitor of...
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Formování sestřihových snRNP v buněčném jádře
Novotný, Ivan ; Staněk, David (advisor) ; Cmarko, Dušan (referee) ; Forstová, Jitka (referee)
1 ABSTRACT There are many structures, suborganelles and bodies in the eukaryotic cell nucleus. These domains provide the nucleus with many specific functions. Nucleolus is specialized compartment serves to ribosomes assembly, nuclear speckles or Splicing Factors Compartment play an important role in RNA processing and best studied of them, Cajal bodies (CBs), are involved in snRNP maturation. However, non-membrane substructures are not unique for cell nucleus; processing bodies (P bodies) found in the cytoplasm are proposed to be important places in mRNA degradation pathway. This work is a compilation of four projects focused on non-membrane cellular bodies; namely, nuclear CBs and cytoplasmic P bodies. Both CBs and P bodies are dynamic structures that continuously exchange their components with surrounding environment. In addition to a widely accepted role of CBs in snRNP biogenesis, we show that the CB serves as a place where snRNPs are regenerated after each round of splicing. Thus, CBs are important nuclear compartment involved in snRNP recycling. To further characterize tri-snRNP assembly in CBs we applied kinetic experiments combined with mathematical modeling and created a kinetic model of tri- snRNP formation in the CB that determined kinetic parameters of tri-snRNP formation. Moreover, our kinetic...
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Replication and transcription of nucleolar DNA
Flusser, Michal ; Smirnov, Evgeny (advisor) ; Staněk, David (referee)
The nucleolus is the most prominent compartment of the cell nucleus and is the place where ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins. Although the nucleolus has been studied for decades its structural and functional organization is still unclear. In particular, the role of various types of DNA participating in the formation of nucleoli along with ribosomal genes is not understood. The present thesis summarizes the current knowledge and views on the nucleolus, focusing on the two synthetic processes, replication and transcription, in mammalian cells. Specific features of these processes in the context of nucleolar ultrastructure remains an unsolved problem of the modern molecular biology.
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Factors important for Cajal body formation
Roithová, Adriana ; Staněk, David (advisor) ; Valentová, Anna (referee)
This research describes the structure and function of nuclear domains called Cajal bodies (CB). CB contain proteins and factors involved in assembly and modification of snRNPs. These bodies are found in vertebrates and invertebrates and even plants. Not all cell types contain CB. Their number and size depends on the transcription activity of cell and cell cycle phase. This paper discusses the factors that affect the CB formation. One of the most important factors is the level of snRNPs and transcription activity. Recently shows that an important role in CB formation has coilin and other components phosphorylation. Other works show the influence of the environment. There is also discussion regulation of CB biogenesis, witch is not yet fully understood. Key words: Cajal bodies, coilin, cell nucleus, snRNP, pre-mRNA splicing, transcription
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The role of acetylation in the RNA recognition motif of SRSF5 protein
Icha, Jaroslav ; Staněk, David (advisor) ; Šenigl, Filip (referee)
Acetylation is emerging as an important posttranslational modification, which is found in thousands of proteins in eukaryotes, as well as prokaryotes. Global proteomic studies implicated acetylation in regulation of various processes like metabolism, gene expression, cell cycle or aging to name a few. In this work I set out to investigate the role of acetylation of a splicing regulatory protein SRSF5 by creating mutations in its acetylation site. I tested the hypothesis that acetylation influences SRSF5 interaction with RNA. I expressed acetylation-mimicking (Q) or non-acetylable (R) mutant of SRSF5 in HeLa cells and measured their interaction with RNA by RNA immunoprecipitation or in vitro by fluorescence anisotropy. Both approaches agreed that mutants interact with RNA less than the wild type protein and Q mutant bound RNA weaker than R mutant. I did not detect further difference in localization or dynamics among the proteins in vivo, which suggests that difference caused by weakened interaction of mutants with RNA was outweighed by other factors influencing SRSF5 behaviour, probably protein-protein interactions. I also found out that mutant SRSF5 proteins do not have a dominant effect on splicing of fibronectin alternative EDB exon. The data obtained give an indirect evidence for the hypothesis that...
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Structure and characteristics of mutant hPrp31 in Retinitis pigmentosa
Těšina, Petr ; Staněk, David (advisor) ; Lišková, Petra (referee)
Retinitis pigmentosa is a hereditary eye disease causing progressive loss of photoreceptor cells, which leads to an irreversible sight handicap and eventually complete blindness. It is a major cause of visual handicap or blindness with very heterogenous genetic background. There are four genes accounting for retinitis pigmentosa that encode splicing factors necessary for spliceosomal assembly and function. Unlike any of the other known genes associated with this disease, these are all expressed ubiquitously throughout the human body. Intriguingly, the mutant forms of these vital splicing factors cause cell-type specific disease affecting only photoreceptor cones and rods. Molecular mechanisms underlying this cell-type specific effect remain elusive. One of these splicing factors is the hPrp31 protein. Its mutant form known as AD29 is the focal point of this thesis. Some of the effects of this mutation on the cellular level have been discovered recently. A creation of the expression vector followed by expression and purification of the truncated hPrp31 protein carrying the AD29 mutation is presented in this thesis. The purified product has been used for production of a αhPrp31 polyclonal rabbit antibody, whose applicability to western blot and immunofluorescence staining has been verified. Moreover...
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