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Role of the SMN complex in snRNP biogenesis
Hanušková, Klára ; Staněk, David (advisor) ; Šubrtová, Adriána (referee)
Small nuclear ribonucleoprotein particles (snRNPs) are an essential component of the spliceosome, a dynamic protein complex that mediates RNA splicing. These particles are composed of a single small nuclear RNA, from which they take their name, a ring of seven Sm or LSm proteins and other accessory proteins. All snRNPs undergo a complex maturation pathway, taking place in the nucleus and cytoplasm of the cell. One important factor in snRNP biogenesis is the SMN complex, which is composed of the SMN, Gemin2-8 and Unrip proteins. Its most important function is to mediate the assembly of the Sm core domain in the cytoplasm of the cell, with the help of the PRMT5 complex. Furthermore, the SMN complex is also involved in snRNA modification, import of the snRNP core, or assembly of the spliceosome itself. Despite more than two decades of research, some of the functions of the SMN complex and its particles have not been fully revealed, and thus it is not clear from the cell to what extent this complex influences not only snRNP biogenesis but also the entire spliceosome. The aim of this work, according to the available research, is to describe the SMN complex in more depth and to understand its functions in snRNP particle biogenesis. Key words: SMN complex, snRNP, snRNA, Sm protein, spliceosome
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Defects of pre-mRNA splicing causing retitinis pigmentosa
Pakhomova, Yelyzaveta ; Staněk, David (advisor) ; Vaňková Hausnerová, Viola (referee)
Retinitis pigmentosa is a genetic disorder affecting the retina. The progression of the disease leads to vision loss. This thesis concentrates on the causation of autosomal dominant retinitis pigmentosa. More specifically, the second biggest responsible mutation group is outlined. The above mentioned gene-mutations group is responsible for the formation of mutant variants of their corresponding splicing proteins. These proteins and consequences of their mutations are reviewed and presented in the thesis. The outline of mutation impact on the retina is presented for each mutated protein. The proteins in question are: PRPF8, PRPF31, PRPF3, PRPF4, PRPF6, SNRNP200, DHX38 (an exemption causing an autosomal recessive retinitis pigmentosa), PAP-1, CWC27 (an exemption causing an autosomal recessive retinitis pigmentosa). The literature review allowed the thesis to conclude that splicing proteins are highly likely to play a critical role in retina's health. In addition, some other noteworthy findings are briefly presented. For example, findings regarding lack of data about some of the mutations. Another example of such finding is that it still remains unknown why these mutations cause such a tissue-specific phenotype. Key words: splicing, retinitis pigmentosa, snRNP, retina, autosomal dominant retinitis...
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Rough Sets on State Spaces of Automata
Staněk, David
This paper discussed a subclass of finite automata, which have ordering on the state sets created by a transition (or next-state) function. Hence, there do not exist cycles of more than one element. We discuss a relation of equality of upper closure on the systems of all subsets of state systems of quasi-automata, which creates an equivalence.
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A role of the 5' cap in Sm-class snRNA biogenesis
Petržílková, Hana ; Staněk, David (advisor) ; Abrhámová, Kateřina (referee)
Small nuclear RNAs (snRNAs) are the core component of the spliceosome which catalyzes pre-mRNA splicing. They undergo a complex biogenesis process which includes snRNA modifications and their assembly into ribonucleoprotein splicing particles snRNPs. The snRNA biogenesis pathway also includes several quality control steps, which block defective snRNPs from entering the spliceosome. One of the important feature involved in Sm-class snRNA quality control is the 5' trimethylguanosine cap (TMG). The capping of Sm-class snRNAs is also connected to the modifications of the first transcribed nucleotide, adenosine, which gets co-transcriptionally 2'-O-methylated and N6-methylated. However, the N6-methylation is later removed by FTO demethylase. Here I shed more light on functional relevance of the various modifications of snRNA 5' cap. I show that the N6-demethylation of the cap is important for normal metabolism of snRNAs, especially U2 snRNA. Next, I provide evidence that the 5' cap plays a role in quality control of 5' truncated snRNAs. Our data suggest that the truncated snRNAs accumulate immature monomethylated caps and are bound by a specific cap binding complex IFIT1/2/3. I propose that the 5' truncated snRNAs are partially stalled in early stages of snRNA biogenesis and are targeted by quality...
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Non-coding RNAs in oocyte and early embryo
Aleshkina, Daria ; Šušor, Andrej (advisor) ; Staněk, David (referee) ; Krylov, Vladimír (referee)
Once considered as 'transcriptional noise' noncoding RNAs (ncRNAs) nowadays are known to be key molecules in major cellular processes. NcRNAs are expressed at very high levels as only 2% of transcribed genome corresponds to protein-coding RNAs in higher eukaryotes. Various ncRNAs are known to have structural, functional, or regulatory roles, but the influence of the majority of non-coding transcripts is still unclear. Among ncRNAs, long ncRNAs (lncRNAs, longer than 200 bp) are of particular interest. LncRNAs do not have a uniform function but many studies observed lncRNA-based regulations at the transcriptional and translational levels. Therefore, novel lncRNAs could specifically fine-tune protein synthesis in the highly differentiated cell types. Particularly, fully-grown mammalian oocyte and early embryo require precisely controlled translation of maternal transcripts to coordinate meiotic progression and early embryo development while transcription is silent. We aimed to study the involvement of ncRNAs in protein synthesis and consequent influence on the oocyte and early embryo physiology. For the first time, we analysed the expression and distribution of several ncRNAs, namely Brain cytoplasmic RNA 1 (BC1), lncRNA in Oocyte Specifically Expressed (Rose), RNA Component of 7SK Nuclear...
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Appeals in criminal proceedings
Staněk, David ; Vokoun, Rudolf (advisor) ; Bohuslav, Lukáš (referee)
APPELS IN CRIMINAL PROCEEDINGS ABSTRACT The goal of this Diploma thesis is to analyse the legal regulation of appeals in criminal proceedings and the issue of this regular remedial measure in general. Author's goal is to present readers with the historical genesis of the institution of appeal since early feudalism until the adoption of current Criminal Procedure Code, considering the most important amendments of this law, and to put the intrastate legal regulation into the context of international law and embedment of the right of appeal in criminal proceedings in international treatises. The author also aims to explain appeal as regular remedial measure together with other remedial measures in Criminal Procedure Code and to introduce basic and specific principles, that impact the legal regulation. In the main chapter the recent regulation of appeals in Criminal Procedure Code is critically analysed. The author further deals with the admissibility and effects of the appeal, by all parties that are legitimate to appeal, the time limit, place and means of filling it, while listing practical examples of abnegation of the right to appeal to a higher court and withdrawal of the appeal, content requirements and possible problems in practice, the operation of the court of first instance after appealing, and also...
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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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Factors important for Cajal body formation
Roithová, Adriana ; Staněk, David (advisor) ; Valentová, Anna (referee)
This research describes the structure and function of nuclear domains called Cajal bodies (CB). CB contain proteins and factors involved in assembly and modification of snRNPs. These bodies are found in vertebrates and invertebrates and even plants. Not all cell types contain CB. Their number and size depends on the transcription activity of cell and cell cycle phase. This paper discusses the factors that affect the CB formation. One of the most important factors is the level of snRNPs and transcription activity. Recently shows that an important role in CB formation has coilin and other components phosphorylation. Other works show the influence of the environment. There is also discussion regulation of CB biogenesis, witch is not yet fully understood. Key words: Cajal bodies, coilin, cell nucleus, snRNP, pre-mRNA splicing, transcription
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Spliceosome assembly
Hausnerová, Viola ; Staněk, David (advisor) ; Chalupníková, Kateřina (referee)
Pre-mRNA splicing is a process in which introns are removed from eukaryotic transcripts and exons are ligated together. Splicing is catalyzed by spliceosome, a large ribonucleoprotein complex composed of five small nuclear RNAs and more than 100 additional proteins, which recognizes 5' splice site, branch point site and 3' splice site and performs two transesterification reactions to produce mRNA molecules. 5' splice site is recognized by U1 snRNP and U2 auxiliary factor (U2AF) is involved in branch point and 3' splice site recognition in the early splicing complex. There is some evidence of splice sites cooperation during intron recognition in vitro but little is known about the situation in vivo. Using Fluorescence resonance energy transfer (FRET) and RNA immunoprecipitation (RIP) methods, we have investigated the early stages of spliceosome assembly. We have employed splicing reporters based on -globin gene and MS2 stem loops to detect interactions of proteins on RNA molecule directly in the cell nucleus. Results of FRET indicate that intact 5' splice site is required for U2AF35 interaction with 3' splice site and that U1C recruitment to 5' splice site is partially limited upon 3' splice site mutation. We have also confirmed by RIP that U2 snRNP association with pre-mRNA molecule requires presence of 5'...
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