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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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To cap or not to cap? Eukaryotic translation initiation with a special interest in human opportunistic pathogen C. albicans
Feketová, Zuzana ; Pospíšek, Martin (advisor) ; Půta, František (referee) ; Vanáčová, Štěpánka (referee)
Candida albicans belongs to serious human opportunistic pathogens, causing severe health complications to immunocompromised patients. To my best knowledge, it is the only organism that survives with unmethylated cap structures found on the 5'ends of mRNA molecules. Using functional assay, I demonstrated that orf19.7626 codes for C. albicans translation initiation factor 4E (Ca4E). We couldn't prove our hypothesis, that Ca4E could be responsible for the unmethylated cap recognition in our model organism S. cerevisiae. Candida sp. possesses also another rather unusual feature - ambiguous CUG codon. In most of the cases, CUG is decoded as a serine, but sometimes also as a leucine. This gives rise to a so called "statistical proteome". One CUG codon is also part of the mRNA coding for Ca4E protein, therefore two versions of Ca4E-Ca4ELeu and Ca4ESer -might occur in C. albicans simultaneously. Both of them are able to rescue deletion of S. cerevisiae eIF4E gene, but they confer temperature sensitivity to the heterologous host. This phenotype is more pronounced with the Ca4ELeu version. We observed milder temperature sensitive phenotype after co-expression of Ca4E together with C. albicans eIF4G (Ca4G). Conformational coupling between eIF4E and eIF4G leads to enhanced affinity of eIF4E to the cap...
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Role of RecQ helicases in maintenance of genomic stability during mitosis
Černoch, Marek ; Janščák, Pavel (advisor) ; Půta, František (referee)
Helicases are proteins capable of unwinding nucleic acids, their malfunction can be dangerous for genome stability of the cell. Five RecQ-family helicases identified in human cells participate in many cellular events during the whole cell cycle, including mitosis, and therefore are very important for correct functioning. The mutations in RecQ helicases can cause them to malfunction and seriously damage various cell processes, for example DNA replication, DNA damage control or sister chromatids separation. The mutations can also lead to dangerous syndromes, with the hallmark symptom of increased risk of cancer.
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The function of Slu7 protein in Saccharomyces cerevisiae pre-mRNA splicing
Ničová, Eva ; Půta, František (advisor) ; Vašicová, Pavla (referee)
Alternative splicing is one of the mechanisms how to regulate gene expression. Under different conditions, different mRNAs encoding proteins with different function, localization or stability can be made from one cellular transcript. The human hSlu7 protein affects the alternative splicing of some genes through alternative 3'splice site (3'SS) selection. Although it was thought that alternative splicing is absent from Saccharomyces cerevisiae, recent results argue against such conclusion. We therefore decided to characterize the function of the yeast Slu7 protein, which participates in the second step of splicing and is closely associated with the 3'SS selection. We focused on a highly conserved uncharacterized motif in the essential part of the Slu7 protein named the RED motif. Mutations in this motif caused second step splicing defects with some substrates and altered the alternative 3'SS usage ratio of some splicing constructs. Our results implicate a role for the RED motif in selecting proper 3'splice sites, especially the distal ones. Genetic interactions of slu7 mutations with PRP22 and PRP45 mutant alelles add to the intricate interaction network of splicing factors and suggest a possible role of Slu7p in facilitating the Prp22p association with the spliceosome.
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Exosome and its role in RNA metabolism of budding yeast S. cerevisiae
Holická, Eliška ; Půta, František (advisor) ; Groušl, Tomáš (referee)
Exosome is a protein complex present in the yeast nucleus and cytoplasm, which participates in RNA degradation, processing and turnover. The core of exosome consists of nine catalytically inactive subunits, which physically associate with RNA nuclease Rrp44. The function of exosome is dependent on many cofactors or facultatively associated enzymes, and these associations provide high versatility of the complex. In different compartments the complex works by other means and plays a role in distinct processes. In nucleus, exosome acts mainly in pre-RNA processing, whereas in cytoplasm its major role is to degrade native mRNA. Nevertheless, in all of these processes, its general role is the 3' exonucleolytic cleavage of single-stranded RNA. Exosome has homologs in many various kinds of organisms - e. g. different types of bacterial nucleases, archeal exosome, human PM-Scl complex (or exosome), which implicates high conservation of this degradation machinery. Thus, it is very likely that some exosome components lost their original function over the evolution, more than that the yeast exosome is an evolutionary innovation.
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Splicing of atypical introns in S. cerevisiae
Cit, Zdeněk ; Půta, František (advisor) ; Pichová, Alena (referee)
Pre-mRNA splicing is a vital process of gene expression important for all eukaryotic organisms. For the proper function of this very complex and dynamic event the presence of few specialized RNA and many proteins that hold a variety of tasks is necessary, not only inside the splicing complex itself, but also beyond this complex. The Prp45 is one of the proteins involved in pre-mRNA splicing in yeast Saccharomyces cerevisiae. Its human homologue, SNW1/SKIP, is involved in splicing but also in other crucial cell processes. The Prp45 protein was reliably reported only to participate in the second transesterification reaction of splicing. But there are also data suggesting its possible involvement in the first transesterification reaction. This work provides further evidences linking protein Prp45 with the first splicing reaction, obtained by the research of cells carrying the mutant allele prp45(1-169). Cells carrying this allele show dropped splicing and accumulation of pre-mRNAs. This thesis therefore also investigated the possible influence of Prp45 protein on the RNA export from the nucleus to the cytoplasm. But no connection between this protein and RNA transport was discovered. Keywords pre-mRNA splicing; Saccharomyces cerevisiae; Prp45; Mer1; Mud2; Prp22; Rrp6; AMA1; SNW1/SKIP
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The role of cyclophilins in splicing
Cit, Zdeněk ; Půta, František (advisor) ; Novotný, Ivan (referee)
Splicing is a process essential for the proper functioning of eukaryotic cells. It is a complicated and highly dynamic process, participated by large numbers of proteins which perform diverse functions, either directly within the splicing complex, or sometimes outside of it. Among the proteins, which play an important role in splicing, are cyclophilins. Cyclophilins probably cause conformational changes in the other components of splicing complex. They can also maintain them in the desire conformation thereby they contribute to the dynamics of the spliceosome. This work provides an overview of cyclophilins, which were confirmed to participate on pre-mRNA splicing, and summarizes suggestions of possible functions that these proteins may perform.
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The interaction of Prp22 and Prp45 proteins in budding yeast spliceosome
Senohrábková, Lenka ; Půta, František (advisor) ; Malcová, Ivana (referee)
Protein Prp22 is a DEAH box RNA helicase, which plays two distinct roles in pre-mRNA splicing: it participates in second transesterification step (ATP independent function) and it releases mature mRNA from the spliceosome (ATP dependent function). Prp45p, yeast ortholog of the human transcription co-regulator SNW/SKIP, is an essential splicing factor, it is included in spliceosome throughout the splicing reaction. Mutant prp45(1-169) genetically interacts with some alleles of NTC complex and second step splicing factors, one of them is also gene PRP22. Here we present, that mutants prp22(-158T) and prp22(-327A), which are synthetically lethal with prp45(1-169), express lower amount of Prp22p due to the mutation in upstream regulation region. Mutants prp22(-158T), prp22(300PPI) and prp22(-327A) affect splicing of pre-mRNA with mutation in 5'ss with respect to sequence of the second exon. N-terminal mutants prp22(∆301) and prp22(∆350) are synthetically lethal with prp45(1-169). Synthetic lethality is possibly caused by lower efficiency of Prp22 recruitment to the spliceosomes, which is no more viable for cells.
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DNA-binding properties of the CSL proteins of Schizosaccharomyces pombe
Ptáčková, Martina ; Půta, František (advisor) ; Doležal, Pavel (referee)
As the effector component of the Notch signaling pathway the transcription factors of the CSL family (CBF1/RBP-Jκ/Suppressor of Hairless/Lag-1) are essential for many developmental processes in metazoan organisms, but they can function also independently of Notch. Recently, their presence was proved in fungal organisms lacking the Notch pathway as well as most of the known metazoan interacting partners. Cbf11 and Cbf12, the CSL proteins of the unicellular yeast Schizosaccharomyces pombe, were determined experimentally as non-essential nuclear transcription factors, which regulate cell adhesion, extracellular material production, colony morphology, septation and daughter cell separation, coordination of nuclear and cell division, and ploidy maintenance in an antagonistic way. The responsive genes of these factors are not known yet. In this study, genes of S. pombe, whose promoter regions represent potential direct targets for the Cbf proteins binding, were predicted. The binding of the Cbf11 and Cbf12 proteins, and of a truncated version Cbf12∆N to CSL response elements contained in the regulatory regions of selected S. pombe genes was tested in vitro by EMSA, and consequently, in the case of the Cbf11 protein, also in vivo by ChIP. Cbf11 and Cbf12∆N recognize specifically the response elements in...
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The TUB3 intron splicing in PRP45 mutant cells
Konířová, Jana ; Půta, František (advisor) ; Zimmermannová, Olga (referee)
Protein Prp45, an essential factor of the yeast Saccharomyces cerevisiae, is implicated in pre-mRNA splicing. A truncated version of the PRP45 gene, prp45(1-169), which exhibits a temperature sensitivity, was previously prepared in our laboratory. The aim of this work is to contribute to better understanding of prp45(1-169) mutant phenotype. We tested the prp45(1-169) strain for its response to microtubule inhibitor benomyl and then we found that TUB3 overexpression from plasmid rescues discovered prp45(1-169) mutant cells hypersensitivity to benomyl. In addition, we studied the influence of TUB1, TUB3, and COF1 intron deletion on prp45(1-169) strain temperature sensitivity. Using RT-qPCR method we found that prp45(1-169) mutation results in the distinctive increase of pre-mRNA level for all tested genes, that could implicate that pre-mRNA splicing in these cells is affected before first transesterication.
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