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The elucidation of the causes of neurogenetic diseases by the MPS data analysis using advanced algorithms
Staněk, David ; Laššuthová, Petra (advisor) ; Halbhuber, Zbyněk (referee) ; Kemlink, David (referee)
8 Summary The thesis "The elucidation of the causes of neurogenetic diseases by the MPS data analysis using advanced algorithms" is focused on processing the massively parallel sequencing (MPS) data from a gene panel, whole-exome sequencing (WES) and whole-genome sequencing (WGS). The aim of the study was to develop a suitable pipeline to evaluate at least 250 MPS gene panel data, 150 WES data and 20 WGS data in order to improve molecular genetic testing of rare neurogenetic disorders. Associated data management and database implementation is also described. Targeted gene panel sequencing A custom-designed gene panel consisting of ge- nes previously associated with the disease was used. In the Epileptic Encephalopathy (EE) panel, two prerequisites need to be met for inclusion into the panel: the gene has to have been published in at least two independent publications OR at least in one publication but in multiple independent families. In the case of the EE panel, 112 genes were included. The targeted gene panel sequencing was then performed on 257 patients with EE. Pathogenic or likely pathogenic (according to ACMG criteria) variants have been found in 28% of patients (72 out of 257). Further analysis of the pathogenic or likely pathogenic variants was performed (76 in total); the variants were grouped by...
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Influence of transcription regulatory elemets on pre-mRNA splicing
Volek, Martin ; Staněk, David (advisor) ; Malík, Radek (referee)
In the process of pre-mRNA splicing introns are removed from pre-mRNA and exons are joined together. Current studies show, that about 95 % of genes, which contain more than two exons, can undergo alternative splicing. In this process some exons are included in or excluded from the final mRNA. Majority of pre-mRNA splicing take place co- transcriptionaly at this time RNA polymerase II is still attached to pre-mRNA. Alternative splicing is complex process that takes place in a close proximity of DNA and histones that might modulate alternative splicing decisions. Futher studies have validated fibronectin gene (FN1) and his alternative exons EDA and EDB (extra domain A and B) as suitably model for studying alternative splicing. Study using FN1 minigene reporter system, which is composed from EDA exon and two surrounding introns and exons, has proved that insertion of transcription enhancer SV40 infront of promotor, the level of EDA inclusion is decreased. So far, has not been prooved if this mechanism can function in real genome context and if distal transcription elements can influence alternative splicing. In this study, we have predicted transcription enhancer for FN1 gene by using The Ensemble Regulatory Build and FANTOM 5. The predicted transcription enhancer, is located 23,5 kbp upstream of TSS...
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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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