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Functional analysis of the TSSC4 chaperone during snRNP formation
Vojáčková, Jitka ; Staněk, David (advisor) ; Vaňková Hausnerová, Viola (referee)
Splicing is a process, during which non-coding sequences (introns) are cleaved out of pre-mRNA, and exons are ligated. This whole process is catalysed by a multi-megadalton splicing complex, composed of five small nuclear ribonucleoprotein particles (shortly snRNPs), which each contains its own small nuclear RNA molecule and specific set of proteins. During the biogenesis of snRNPs, U4 and U6 snRNPs are assembled to form the di-snRNP, which further associates with U5 snRNP and gives rise to tri-snRNP. With the help of mass spectrometry, we have found previously uncharacterized protein interacting with U5 snRNP, called TSSC4. By immunoprecipitation, I confirmed TSSC4 as a U5 snRNP specific protein and identified the region of TSSC4 responsible for interaction with U5 snRNP. I also showed that TSSC4 interacts with PRPF19, a component of complex driving the catalytic activation of the spliceosome and that this interaction is U5 snRNP-independent. Knockdown of TSSC4 in HeLa cells results in accumulation of di-snRNAs and U5 snRNP in Cajal bodies, nuclear compartments involved in snRNP biogenesis. Similar phenotype was previously observed upon inhibition of tri-snRNP assembly. To analyse the importance of TSSC4 for tri-snRNP assembly, I separated individual snRNPs by glycerol gradient ultracentrifugation...
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Functional analysis of the TSSC4 chaperone during snRNP formation
Vojáčková, Jitka ; Staněk, David (advisor) ; Vaňková Hausnerová, Viola (referee)
Splicing is a process, during which non-coding sequences (introns) are cleaved out of pre-mRNA, and exons are ligated. This whole process is catalysed by a multi-megadalton splicing complex, composed of five small nuclear ribonucleoprotein particles (shortly snRNPs), which each contains its own small nuclear RNA molecule and specific set of proteins. During the biogenesis of snRNPs, U4 and U6 snRNPs are assembled to form the di-snRNP, which further associates with U5 snRNP and gives rise to tri-snRNP. With the help of mass spectrometry, we have found previously uncharacterized protein interacting with U5 snRNP, called TSSC4. By immunoprecipitation, I confirmed TSSC4 as a U5 snRNP specific protein and identified the region of TSSC4 responsible for interaction with U5 snRNP. I also showed that TSSC4 interacts with PRPF19, a component of complex driving the catalytic activation of the spliceosome and that this interaction is U5 snRNP-independent. Knockdown of TSSC4 in HeLa cells results in accumulation of di-snRNAs and U5 snRNP in Cajal bodies, nuclear compartments involved in snRNP biogenesis. Similar phenotype was previously observed upon inhibition of tri-snRNP assembly. To analyse the importance of TSSC4 for tri-snRNP assembly, I separated individual snRNPs by glycerol gradient ultracentrifugation...
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Gene regulation in four dimensions
Vaňková Hausnerová, Viola ; Lanctôt, Christian (advisor) ; Převorovský, Martin (referee) ; Krásný, Libor (referee)
Transcription has turned out to be a discontinuous process when imaged at a single cell level. This observation is referred to as transcriptional bursting or pulsing and has been detected in a variety of organisms ranging from bacteria to mammalian cells. The dynamics of transcriptional pulsing are influenced by the properties intrinsic to the transcriptional process, as well as by upstream factors: chromatin environment, signalling molecules, cell cycle stage etc. In the first part of this thesis, we focused on the regulation of transcriptional pulsing in the nucleolus. Using imaging of living cells, we detected pulsatile transcription of a transgene with nucleolar localization whose expression was mediated by RNA polymerase II. In the second part of the thesis, we investigated the relationship between chromatin decondensation and transcriptional dynamics. We used hyperosmotic medium to induce global condensation of chromatin and revealed that upon chromatin decondensation, a transient spike in transcriptional intensity occurs in induvial living cells. Next, we analysed expression of TFRC and POLR2A genes in several cell cycle stages using single molecule RNA FISH. We detected increase in both frequency and size of transcriptional pulses during a limited time window which coincided with chromatin...
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Promoter-terminator interactions in eukaryotic RNA polymerase II transcription
Petr, Martin ; Hozák, Pavel (advisor) ; Vaňková Hausnerová, Viola (referee)
Gene loops are chromatin structures formed by juxtaposition of distal genomic regions. Since these regions are often involved in transcription cycle control, gene loops therefore provide another mechanism of regulation of gene expression. This thesis summarizes recent findings about gene loops, focusing specifically on loops formed by interactions between promoter and terminator regions of genes transcribed by the eukaryotic RNA polymerase II. Different cases of gene loops discovered in several yeast genes, the mammalian BRCA1 tumor suppressor and the HIV-1 integrated provirus are described, including mechanisms that possibly lead to the formation of these structures. Since gene loops and interactions between promoter and terminator in yeast have been linked to the transcriptional memory, their involvement in this phenomenon is discussed. Finally, as BRCA1 and HIV-1 are directly linked to serious human diseases, the potential significance of alterations of gene loops in the development of various pathological conditions is presented. Keywords: gene loops, chromatin loops, chromatin conformation, transcriptional memory, transcription, gene expression, regulation
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