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Analysis of co-transcriptional spliceosome assembly on RPL22B
Hájková, Karolína ; Abrhámová, Kateřina (advisor) ; Cvačková, Zuzana (referee)
In eukaryotic organisms, the process of splicing is used to remove non-coding sequences - introns - from transcripts to form mRNA, and together with transcription, it also allows to regulate the amount of the resulting RNA that will be used further in translation. The protein of interest in our study is Rpl22, which binds the intron of its own transcript and its paralog and can inhibit their splicing. This thesis examines the mechanism by which the inhibition occurs and provides new information about the regulation of RPL22 transcript expression via its intron. Using co-transcriptional spliceosome assembly analysis on the RPL22B transcript, we were able to observe that the recognition of splice sites by the U1 snRNP, Msl5 and Mud2, is not affected by the inhibition. Thus, the inhibition of RPL22B splicing will occur later during spliceosome assembly or during spliceosome activation, before the first step of splicing takes place.
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Functional analysis of hPrp8 mutations linked to retinitis pigmentosa.
Matějů, Daniel ; Cvačková, Zuzana (advisor) ; Král, Vlastimil (referee)
hPrp8 is an essential pre-mRNA splicing factor. This highly conserved protein is a component of the U5 small ribonucleoprotein particle (U5 snRNP), which constitutes one of the building blocks of the spliceosome. hPrp8 acts as a key regulator of spliceosome activation and interacts directly with U5 snRNA and with the regions of pre-mRNA that are involved in the transesterification reactions during splicing. Mutations in hPrp8 have been shown to cause an autosomal dominant form of retinitis pigmentosa (RP), an inherited disease leading to progressive degeneration of retina. In this study, we analyzed the effects of the RP-associated mutations on the function of hPrp8. Using BAC recombineering, we created mutant variants of hPrp8-GFP construct and we generated stable cell lines expressing the recombinant proteins. The mutant proteins were expressed and localized to the nucleus. However, one of the missense mutations affected the localization and stability of hPrp8. Further experiments suggested that RP-associated mutations affect the ability of hPrp8 to interact with other components of the U5 snRNP and with pre-mRNA. We further studied the biogenesis of U5 snRNP. We depleted hPrp8 by siRNA to interfere with U5 snRNP assembly and we observed that the incompletely assembled U5 snRNPs accumulate in...
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Changes in domain organization of the plasma membrane in the stress response
Vaškovičová, Katarína ; Malínský, Jan (advisor) ; Zimmermannová, Olga (referee) ; Cvačková, Zuzana (referee)
MCC/eisosomes are yeast plasma membrane microdomains that respond to changes in both extracellular and intracellular conditions and activate important stress-related signaling pathways. In this study, we investigated the function of MCC/eisosomes under the conditions of chronic glucose depletion. We found that MCC/eisosomes regulate mRNA decay under these conditions. Specifically, we demonstrated that the sequestration of the evolutionarily conserved Xrn1 exoribonuclease at MCC/eisosomes leads to the attenuation of its enzymatic activity. Modulation of activity by the enzyme localization may represent a novel and effective mechanism in regulation of biochemical pathways. Moreover, our results suggested that an MCC protein Nce102 might play a role in vacuolar fusion and lipid droplets degradation. We demonstrated that prolonged chronic glucose depletion induces the translocation of Nce102 from MCC to sterol-enriched microdomains in the vacuolar membrane. Deletion mutants lacking Nce102 and its functional homologue Fhn1 exhibited significant delay in vacuole maturation and in turnover of a lipid droplet marker Erg6. The function of MCC/eisosomes in the stress response have been demonstrated in many fungal species. Similar to the microdomain function, also individual protein components of...
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Domain 1.1 of the primary sigma factor and a new expression system for Bacillus subtilis RNA polymerase.
Kálalová, Debora ; Krásný, Libor (advisor) ; Cvačková, Zuzana (referee)
RNA polymerase (RNAP) is a key multi-subunit enzyme of gene expression that, together with the σ factor, forms a holoenzyme and transcribes genetic information from DNA to RNA. RNAP from Bacillus subtilis and its primary factor σA were studied in this thesis. The σA factor determines the specificity for the promoters to which the holoenzyme binds. Part of its structure is domain 1.1, which is likely to prevent binding of σA to the promoter by itself (unless it is part of the holoenzyme) by binding to domains 2 and 4. The first part of the thesis verifies the hypothesis that domain 1.1 binds domains 2 and 4 and thus prevents binding of σA to the promoter. To this end, various domain constructs have been created and their interactions have been tested. Domain interaction was tested by Nitrocellulose Filter Binding Assay, EMSA, and in vitro transcription. The results did not show significant interaction between domains. The second part of the thesis deals with the creation of a tool for the study of the enzymatology of RNAP from B. subtilis - recombinant RNAP (rRNAP). First, a plasmid construct for expression of rRNAP in Escherichia coli was constructed by a series of cloning steps, followed by protein isolation and characterization. Isolation was achieved without contamination by σ factors (this...
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The assembly of perinuclear actin stress fibers and their role in cell movement
Votavová, Barbora ; Vomastek, Tomáš (advisor) ; Cvačková, Zuzana (referee)
Nucleus is the largest cellular organelle in animal cells. Due to its bulky nature and the stiffness of nuclear lamina the nucleus constitutes the substantial problem for migrating cells where nucleus has to move. The actomyosin generated forces and LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, that is composed of SUN and nesprin proteins, play key role in nuclear movement. LINC complex mechanically couples nuclear lamina to the cytoskeleton and allows the forces exerted by the cytoskeleton to move the nucleus. Perinuclear actin fibers, also termed actin cap, mechanically link focal adhesions with nucleus and they may generate forces that position the nucleus in a way that is optimal for cellular movement. However, molecular mechanism of how perinuclear actin fibers and LINC complex orchestrate the nuclear movement and functional significance of this process remain poorly understood. The specific aim was to determine the mechanisms by which perinuclear actin fibers are formed and how are these mechanisms employed to facilitate cell migration. The role of LPA-RhoA signaling axis and LINC complex in the formation of perinuclear actin fibers was also examined. It was confirmed that LPA is essencial stimulus during actin cap formation. On the other hand, FAK kinase was found necessary for...
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Functional analysis of hPrp8 mutations linked to retinitis pigmentosa.
Matějů, Daniel ; Cvačková, Zuzana (advisor) ; Král, Vlastimil (referee)
hPrp8 is an essential pre-mRNA splicing factor. This highly conserved protein is a component of the U5 small ribonucleoprotein particle (U5 snRNP), which constitutes one of the building blocks of the spliceosome. hPrp8 acts as a key regulator of spliceosome activation and interacts directly with U5 snRNA and with the regions of pre-mRNA that are involved in the transesterification reactions during splicing. Mutations in hPrp8 have been shown to cause an autosomal dominant form of retinitis pigmentosa (RP), an inherited disease leading to progressive degeneration of retina. In this study, we analyzed the effects of the RP-associated mutations on the function of hPrp8. Using BAC recombineering, we created mutant variants of hPrp8-GFP construct and we generated stable cell lines expressing the recombinant proteins. The mutant proteins were expressed and localized to the nucleus. However, one of the missense mutations affected the localization and stability of hPrp8. Further experiments suggested that RP-associated mutations affect the ability of hPrp8 to interact with other components of the U5 snRNP and with pre-mRNA. We further studied the biogenesis of U5 snRNP. We depleted hPrp8 by siRNA to interfere with U5 snRNP assembly and we observed that the incompletely assembled U5 snRNPs accumulate in...
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Nucleolus and Its Associated Chromatin
Cvačková, Zuzana ; Raška, Ivan (advisor) ; Smetana, Karel (referee) ; Nedvídek, Josef (referee)
Mammalian chromosomes occupy chromosome territories within nuclear space the positions of which are generally accepted as non-random. However, it is still controversial whether position of chromosome territories is maintained, together with their neighborhood, in daughter cells. We addressed this issue and investigated maintenance of various chromatin regions of unknown composition as well as nucleolus-associated chromatin, a significant part of which is composed of ribosomal genes-bearing chromosomes. The photoconvertible histone H4-Dendra2 was used to label such regions in transfected HepG2 cells, and its position was followed up to next interphase. The distribution of labeled chromatin in daughter cells exhibited a non- random character. However, its distribution in a vast majority of daughter cells extensively differed from the original ones and the labeled nucleolus-associated chromatin differently located into the vicinity of different nucleoli. Therefore, our results were not consistent with a concept of preservation chromatin position. This conclusion was supported by the finding that the numbers of nucleoli significantly differed between the two daughter cells. Our results support a view that while the transfected daughter HepG2 cells maintain some features of the parental cell chromosome...
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