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The analysis of plasticity of cancer cell invasiveness
Merta, Ladislav ; Brábek, Jan (advisor) ; Šindelka, Radek (referee) ; Staněk, David (referee)
The ability of cancer cells to adopt various invasive modes (the plasticity of cancer cell invasiveness) represents a significant obstacle in the treatment of cancer metastasis. Cancer invasiveness involves various modes of migration. Cells can move together (with the preserved intercellular junctions; collective invasiveness) or individually. Within individual invasiveness, we distinguish two principal invasive modes - mesenchymal and amoeboid. The mesenchymal mode of migration is characterized by an elongated shape, proteolytic degradation of the fibres of the extracellular matrix, and the formation of strong contacts with the extracellular matrix. The amoeboid mode of migration is not dependent on proteolytic activity, the cells are characterized by a round shape and increased contractility, which they use to squeeze themselves through the pores of the extracellular matrix. This thesis deals with the analysis of the plasticity of cancer cell invasiveness, specifically the transitions between individual amoeboid and mesenchymal migration modes, in the 3D environment of the collagen gel as a model of extracellular matrix. The work presents models of mesenchymal-to-amoeboid transition (MAT), which include BLM, HT1080 and MDA-MB-231 cell lines, in which MAT is induced by the expression of...
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Effect of SR-like proteins in maturation and transfer of mRNA to the cytoplasm
Hájková, Karolína ; Abrhámová, Kateřina (advisor) ; Staněk, David (referee)
Metazoan SR proteins is a group of proteins involved in splicing. They are RNA-biding proteins characterized by the presence of serine and arginine-rich domain. The yeast, Saccharomyces cerevisiae, encodes genes for proteins that are similar in amino acid composition and structure to the group of SR proteins and are therefore called SR-like proteins. There are three proteins with RS domain in yeast: Npl3, Gbp2 and Hrb1. These proteins have been shown to have many functions, including, in addition to splicing, quality control of spliced mRNAs and their export from the nucleus to the cytoplasm. This Bachelor's thesis will deal with these three yeast proteins and their role in splicing, export and degradation.
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Splicing Factors in the Regulation of Gene Expression - the Relationship Between Splicing and Transcription in Saccharomyces cerevisiae
Hálová, Martina ; Folk, Petr (advisor) ; Pospíšek, Martin (referee) ; Staněk, David (referee)
Transcription and pre-mRNA processing, e.g., splicing, occur at the same place and time in the context of chromatin. A growing amount of evidence supports the hypothesis that these processes are interconnected. Prp45/SKIP is one of the factors which are believed to mediate the interconnection. The human ortholog, SKIP, is known for affecting mRNA formation on the levels of transcription initiation and elongation. Moreover, it interacts with chromatin modifiers and it is a splicing factor, too. The function of the Saccharomyces cerevisiae ortholog, Prp45, has been so far connected only to pre-mRNA splicing. In this work, we characterized the role of Prp45 in splicing and elaborated the results connecting Prp45 to transcription and chromatin modifications. RNA-seq results showed that pre-mRNA is accumulated in prp45(1-169) cells. This accumulation is not caused by the reduced activity of pathways responsible for RNA degradation. The extent of the splicing inefficiency in prp45(1-169) cells did not depend on either the canonicity of the 5' splice site and branch site or the distance between the branch site and the 3' splice site. Using chromatin immunoprecipitation, we found that prp45(1-169) mutation causes delay in U2 snRNP recruitment to assembling spliceosome. This delay transfers to the later...
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m6A RNA methylation in eukaryotic cells
Petržílková, Hana ; Staněk, David (advisor) ; Folk, Petr (referee)
The N6-methylation of adenosine (m6 A) is the most abundant modification in eukaryotic mRNA. This modification is deposited on RNA co-transcriptionally by the methyltransferase complexes and can also be "erased" by specific demethylases. The existence of m6 A demethylases makes the modification reversible and potentially dynamic, therefore, m6 A could have a function in gene expression regulation. Since the discovery of the first m6 A demethylase FTO, the m6 A has become a hot-topic in RNA-biology research. m6 A is found in mRNAs but also in various non-coding RNAs. Analysis of m6 A distribution on mRNAs revealed the enrichment of m6 A in proximity of a stop codon, in 3' UTRs and possibly around 5' and 3' splice-sites. So far two m6 A methyltransferases have been discovered in vertebrates, METTL3/METTL14 complex is the major methyltransferase and METTL16 deposits m6 A just on a specific subset of RNAs. Additionally, two m6 A demethylases are known - FTO and ALKBH5. Finally, members of protein family with a so-called YTH RNA binding domain were identified as m6 A binding proteins. m6 A serves as a signal affecting various steps of RNA metabolism such as mRNA splicing, nuclear export, translation or RNA degradation. Some of the effects are clearly mediated by the m6 A binding proteins, but also other...
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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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Bacterial RNA polymerase and molecules affecting its function
Jirát Matějčková, Jitka ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Staněk, David (referee)
RNA polymerase (RNAP) transcribes DNA into RNA and is the only transcriptional enzyme in bacteria. This key enzyme responds to external and internal signals from the cell, resolves the intensity of transcription of individual genes and thus regulates gene expression. RNAP is not only affected by its own subunits, but also protein factors, small molecules or small RNAs (sRNAs). The aim of this Thesis was to contribute to the understanding of the regulation of the RNAP and to add missing fragments to this broad topic. The first part of this Thesis is focused on the influence of selected proteins (δ, YdeB, GreA) on the sensitivity of RNAP to the concentration of the initiating nucleoside triphosphate ([iNTP]) during transcription initiation in Bacillus subtilis. We showed that δ affects the sensitivity of RNAP to [iNTP] at [iNTP]-sensitive promoters, but not at [iNTP]-insensitive promoters neither in vitro nor in vivo. The δ subunit is essential for cell survival during competition with other strains, because it enables the cell to react immediately to changing conditions. Further we showed that YdeB protein does not bind to RNAP in B. subtilis, and has not shown any effect on transcription in vitro. We found that both, GreA and YdeB proteins (unlike δ subunit) were unable to affect RNAP by [iNTP] at...
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Determinants of the splice site selection in protein-coding and long non-coding RNAs
Krchňáková, Zuzana ; Staněk, David (advisor) ; Svoboda, Petr (referee) ; Blažek, Dalibor (referee)
In my thesis, I focused on several underexplored areas of RNA splicing regulation. In the first part, I analyzed how chromatin and transcription regulatory elements change pre-mRNA splicing. In the second part, I studied why long non-coding RNAs (lncRNAs) are spliced less efficiently than protein-coding mRNAs. Finally, I was testing the importance of intron for the activating function of lncRNAs. It has been shown that chromatin and promoter identity modulate alternative splicing decisions. Here, I tested whether local chromatin and distant genomic elements that influence transcription can also modulate splicing. Using the chromatin modifying enzymes directly targeted to FOSL1 gene by TALE technology, I showed that changes in histone H3K9 methylation affect constitutive splicing. Furthermore, I provide evidence that deletion of transcription enhancer located several kilobases upstream of an alternative exons changes splicing pattern of the alternative exon. Many nascent lncRNAs undergo the same maturation steps as pre-mRNAs of protein- coding genes (PCGs), but they are often poorly spliced. To identify the underlying mechanisms for this phenomenon, we searched for putative splicing inhibitory sequences. Genome-wide analysis of intergenic lncRNAs (lincRNAs) revealed that, in general, they do not...
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A Multistructure Created By Coverings Of A Set
Staňek, David
This paper focuses on a certain construction of a multistructure on the set of all coverings on the universal set U and discusses properties of this multistructure. The construction itself uses a concept dual to the Ends Lemma, called Beginnings Lemma, which is proved in the paper.
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Quality control in snRNP biogenesis
Roithová, Adriana ; Staněk, David (advisor) ; Malínský, Jan (referee) ; Vomastek, Tomáš (referee)
(English) snRNPs are key components of the spliceosome. During their life, they are found in the cytoplasm and also in the nucleus, where carry out their function. There are five major snRNPs named according to RNA they contain U1, U2, U4, U5 and U6. Each snRNP consists from RNA, ring of seven Sm or LSm proteins and additional proteins specific for each snRNP. Their biogenesis starts in the nucleus, where they are transcribed. Then they are transported into the cytoplasm. During their cytoplasmic phase, the SMN complex forms the Sm ring around the specific sequence on snRNA and cap is trimethylated. These two modifications are the signals for reimport of snRNA into the nucleus, where they accumulate in the nuclear structures called Cajal bodies (CBs), where the final maturation steps occur. There are several quality control points during snRNP biogenesis that ensure that only fully assembled particles reach the spliceosome. The first checkpoint is in the nucleus immediately after the transcription, when the export complex is formed. The second checkpoint is in the cytoplasm and proofreads Sm ring assembly. If the Sm ring formation fails, the defective snRNPs are degraded in the cytoplasm by Xrn1 exonuclease. However, it is still unclear, how the cell distinguishes between normal and defective...
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