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Analýza regulace komplexů cytoplazmatických poly(A) polymeráz
Novák, Jakub ; Staněk, David (advisor) ; Hrossová, Dominika (referee)
The regulation of gene expression is achieved at many levels. Chromatin-based gene regulation has been the central focus of many decades of research; however, posttranscriptional control mechanisms are emerging as a fundamental complement to direct protein synthesis. This thesis is focused on a specific mechanism of posttranscriptional control - the translational regulation of mRNAs in the cell cytoplasm. This control is a consequence of the balance between translational repression and activation and hinges on the selective recognition of regulated mRNAs by RNA-binding proteins and their ability to recruit RNA modifying proteins. In this thesis, Caenorhabditis elegans germline was used to study translational control of the germ cell-enriched gene, gld-2. Mutants of known RNA-binding proteins of the PUF and CPB protein families were analyzed by performing Western blots, using anti-GLD-2 antibodies. Yeast 3-Hybrid system was used to identify the cis-regulatory sites in the gld-2 mRNA conferring translational regulation by members of PUF and CPB protein families. Potential autoregulatory loop of gld-2 gene expression was also investigated. This thesis shows that FBF proteins positively regulate expression of gld-2 and bind to a conserved sequence in the 3'UTR of its mRNA. Mutations of gld-2 negatively affect...
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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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Structure and characteristics of mutant hPrp31 in Retinitis pigmentosa
Těšina, Petr ; Staněk, David (advisor) ; Lišková, Petra (referee)
Retinitis pigmentosa is a hereditary eye disease causing progressive loss of photoreceptor cells, which leads to an irreversible sight handicap and eventually complete blindness. It is a major cause of visual handicap or blindness with very heterogenous genetic background. There are four genes accounting for retinitis pigmentosa that encode splicing factors necessary for spliceosomal assembly and function. Unlike any of the other known genes associated with this disease, these are all expressed ubiquitously throughout the human body. Intriguingly, the mutant forms of these vital splicing factors cause cell-type specific disease affecting only photoreceptor cones and rods. Molecular mechanisms underlying this cell-type specific effect remain elusive. One of these splicing factors is the hPrp31 protein. Its mutant form known as AD29 is the focal point of this thesis. Some of the effects of this mutation on the cellular level have been discovered recently. A creation of the expression vector followed by expression and purification of the truncated hPrp31 protein carrying the AD29 mutation is presented in this thesis. The purified product has been used for production of a αhPrp31 polyclonal rabbit antibody, whose applicability to western blot and immunofluorescence staining has been verified. Moreover...
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The role of coilin in snRNP quality control
Kuzmenko, Darya ; Staněk, David (advisor) ; Abrhámová, Kateřina (referee)
Mammalian genes are transcribed as precursors - pre-mRNA. They contain coding sequences (exons) and non-coding sequences (introns). Splicing, a process of cutting out introns and joining exons to generate mature mRNA, is carried out by a spliceosome. The spliceosome consists of five small nuclear ribonucleoprotein (snRNP) particles and numerous associated proteins. Its assembly is a complex process involving a specific nuclear sub-compartment, the Cajal body (CB). Here, we investigate function of the CB scaffold protein, coilin, in snRNP quality control in HeLa cells. Sequestration of immature snRNP in coilin-deficient cells is analysed by fluorescence in situ hybridisation. We show that without coilin the cells are unable to sequester them. Next, we provide evidence that absence of coilin does not sensitise HeLa cells for perturbation in snRNP maturation in terms of cell proliferation. Moreover, coilin deficiency does not result in significant changes in U4, U5 or U6 snRNA steady state levels. Therefore, coilin, and, in this way, Cajal bodies do not become essential under the conditions of strained snRNP biogenesis.
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Molecular mechanism of quality control during snRNP biogenesis
Klimešová, Klára ; Staněk, David (advisor) ; Krásný, Libor (referee) ; Vomastek, Tomáš (referee)
The spliceosome is one of the largest and most dynamic molecular machines in the cell. The central part of the complex is formed by five small nuclear ribonucleoproteins (snRNPs) which are generated in a multi-step biogenesis pathway. Moreover, the snRNPs undergo extensive rearrangements during the splicing and require reassembly after every intron removal. Both de novo assembly and post-splicing recycling of snRNPs are guided and facilitated by specific chaperones. Here, I reveal molecular details of function of two snRNP chaperones, SART3 and TSSC4. While TSSC4 is a previously uncharacterized protein, SART3 has been described before as a U6 snRNP-specific factor which assists in association of U6 and U4 particles into di-snRNP, and is important for the U4/U6 snRNP recycling. However, the mechanism of its function has been unclear. Here, I provide an evidence that SART3 interacts with a post-splicing complex and propose that SART3 could promote its disassembly. Our data further suggest that SART3 binds U6 snRNP already within the post-splicing complex and thus participates in the whole recycling phase of U6 snRNP. Then, I show that TSSC4 is a novel U5 snRNP-specific chaperone which promotes an assembly of U5 and U4/U6 snRNPs into a splicing-competent tri-snRNP particle. We identified...
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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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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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