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RNA polymerase: The "meeting point" of regulatory networks
Wiedermannová, Jana ; Krásný, Libor (advisor) ; Pospíšek, Martin (referee) ; Valášek, Leoš (referee)
Bacterial RNA polymerase (RNAP) is a multisubunit complex essential for transcription of DNA into RNA. As a key enzyme responsible for regulation of gene expression it interprets regulatory signals from the cell and based on these cues RNAP adjusts transcription level of particular genes. This process is affected both by the regular subunits of RNAP as well as other transcription factors (TFs) directly or indirectly interacting with RNAP. The general focus of this Thesis was to extend the knowledge about the complex transcriptional regulatory networks and about the connections between individual pathways. The main specific topic and the main publication of the thesis are focused on the HelD protein, a novel binding partner of RNAP in Bacillus subtilis. We showed that HelD binds between the secondary channel of RNAP and alpha subunits of the core form of the enzyme. We proved that HelD stimulates transcription in an ATP dependent manner by enhancing transcriptional cycling and elongation. We revealed a new connection in the transcription regulatory machinery when we demonstrated that the stimulatory effect of HelD can be amplified by delta, a small subunit of RNAP specific for gram positive (G+) bacteria. Two other publications of the thesis are dealing with the delta subunit. We solved the 3D...
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Vývoj chemických regulátorů drah mikroRNA a RNAi
Bruštíková, Kateřina ; Svoboda, Petr (advisor) ; Bařinka, Cyril (referee) ; Pospíšek, Martin (referee)
MicroRNAs are noncoding RNAs inducing sequence-specific posttranscriptional inhibition of gene expression and represent the major class of small endogenous RNAs in mammalian cells. Over 2,500 of human microRNAs potentially regulating more than 60% of human protein-coding genes have been identified. MicroRNAs participate in the majority of cellular processes, and their expression changes in various diseases, including cancer. Currently, there is no efficient small chemical compound available for the modulation of microRNA pathway activity. At the same time, small chemical compounds represent excellent tools for research of processes involving RNA silencing pathways, for biotechnological applications, and would have a considerable therapeutic potential. The presented work represents a part of a broader project, whose ultimate goal is: (i) to find a set of small molecules allowing for stimulation or inhibition of RNA silencing and (ii) to identify crosstalks between RNA silencing and other cellular pathways. This thesis summarizes results from the first two phases of the project, the development of high-throughput screening assays and the high- throughput screening (HTS) of available libraries of small compounds. To monitor the microRNA pathway activity, we developed and optimized one biochemical...
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Substrate cleavage by mammalian Dicer isoforms
Kubíková, Jana ; Svoboda, Petr (advisor) ; Pospíšek, Martin (referee)
Host organisms evolved antiviral responses, which can recognize the viral infection and deal with it. One of the frequent signs of viral infection in a cell is appearance of double-stranded RNA (dsRNA). One of the pathways responding to dsRNA is RNA interference (RNAi), which functions as the key antiviral defence system in invertebrates and plants. Mammals, however, utilize for antiviral defence a different dsRNA-sensing pathway called the interferon response. RNAi functions only in mammalian oocytes and early embryonal stages although its enzymatic machinery is present in all somatic cells, where it is employed in the microRNA pathway. A previous study indicated that the functionality of RNAi in mouse oocytes functions due to an oocyte-specific isoform of protein Dicer (DicerO ), which is truncated at the N-terminus. In my thesis, I aimed to assess whether DicerO processes RNAi substrates more efficiently in vitro than the full-length Dicer (DicerS ), which is found in somatic cells. Therefore, I developed Dicer purification protocol for obtaining both recombinant mouse Dicer isoforms of high purity. I examined their activity in a non-radioactive cleavage assay using RNA substrates with structural features characteristic of RNAi substrates. My results suggest that recombinant DicerO and DicerS do not...
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Preparation of yeast system for investigation of the human translation initiation
Holásková, Lucie ; Pospíšek, Martin (advisor) ; Cuchalová, Lucie (referee)
Protein synthesis is principally regulated at the initiation stage in which eIF4F complex plays an important role. The eIF4F complex contains three subunits - eIF4A, eIF4E and eIF4G. The eIF4E is cap binding protein, the eIF4A is RNA dependent helicase which unwinds secondary structures at mRNA and scaffolding eIF4G protein. The interaction with other translation initiation factors is important for protein synthesis. The goal of my thesis was to create a new Saccharomyces cerevisiae yeast strain with the human eIF4F factor. Firstly I replaced yeast eIF4E protein with human eIF4E protein. I used a cre/loxP recombination to prepare yeast strains with deleted genes eIF4GI (huΔ4G1) and eIF4GII (huΔ4G2). Characterization of the new yeast strains showed that the human eIF4E protein replaced yeast ortholog factor better than the eIF4E protein from yeast Candida albicans. First experiments showed putative role of the eIF4GII protein during the cell growth under the temperature and osmotic stress. Key words: translation initiation, eIF4E, eIF4G, Saccharomyces cerevisiae
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Impact of the rRNA modifications on protein synthesis
Kročová, Eliška ; Pospíšek, Martin (advisor) ; Holá, Dana (referee)
A ribosome is a supramolecular structure, which mediates synthesis of all cellular proteins, and therefore is essential for cell life. The fact, that some nucleotides of ribosomal RNA are modified, is known for forty years. However only recently, successful deeper studies on how the individual modifications are synthesized and what is their effect on ribosome synthesis and function appear. Some particular nucleotide modifications are important for the ribosome formation (like m1 acp3 Ψ1191 SSU), some others influence proper function of the ribosome (e.g. Um2921, Gm2922, Ψ2923 LSU, m1 acp3 Ψ1191 SSU). Majority of modified nucleotides in eukaryotic rRNA is being recognized by small nucleolar RNA (snoRNA). Few nucleotides is, however, recognized and subsequently modified by specific proteins. These proteins also play crucial role in ribosome maturation. In thesis presented, current knowledge on the role of ribosomal RNA nucleotide modifications during their formation and maturation, and on their function is summarized and overviewed.
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Protein synthesis in cellular stress
Cienciala, Martin ; Pospíšek, Martin (advisor) ; Holá, Dana (referee)
Environmental stress is a daily bread for organisms across many different branches of life. Very complex response mechanisms have evolved to tackle such insults. Yeast Saccharomyces cerevisiae is adapted especially well for counteracting oxidative and osmotic stress. These unfavorable conditions usually lead to inhibition of protein synthesis. The GCN2 kinase is thought to be responsible for this phenomenon. General inhibition of protein synthesis is accompanied by an increase in expression of proteins engaging in stress response. Production of these proteins is often preceded by specialized regulatory processes, that operate on various stages of expression. This thesis will try to present the diversity and complexity of the individual regulatory layers.
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The role of HOG MAPK signaling pathway during osmotic stress in Saccharomyces cerevisiae
Vrbová, Michaela ; Pospíšek, Martin (advisor) ; Janderová, Blanka (referee)
THE ROLE OF HOG MAPK SIGNALING PATHWAY DURING OSMOTIC STRESS IN SACCHAROMYCES CEREVISIAE Budding yeast (Saccharomyces cerevisiae) cells utilize a conserved mitogen-activated protein kinase (MAPK) signaling cascade (the high- osmolarity glycerol or HOG pathway) during conditions of increased external osmolarity. It evokes cellular responses necessary to permit continued growth. Activation of HOG pathway with Hog1 MAP kinase results in production of glycerol to prevent dehydration and up regulation of other Hog1 dependent genes because of cell adaptation. We were trying to find difference in translation between wild-type cells and two mutants of hog1 gene before and after 0,4 M NaCl osmotic stress (2, 6, 30 min). We used deletion mutant hog1Δ and hog1-as mutant with point mutation which allows inhibition of Hog1 MAPK during presence of specific AS inhibitor. We tested AS inhibitor by plate test and have found optimal concentration of 5 μM for blocking Hog1 MAPK in hog1-as mutant. Translation profiling proves that osmotic stress decreases translation in general. Hog1Δ mutant and hog1-as AS inhibited mutant behave similarly and their translation recovers slower than the wild-type's. That confirms that HOG1 gene is important for cell recovery from the osmotic stress. Microarray analysis shows that Hog1 dependent...
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Role of the oncogenic microRNAs miR-17-92 and miR-155 in the regulation of hematopoietic differentiation and leukemogenesis
Pospíšil, Vít ; Stopka, Tomáš (advisor) ; Pospíšek, Martin (referee) ; Machová Poláková, Kateřina (referee)
(English version): Hematopoietic differentiation is highly ordered multistep process, where generation of terminal blood cells is dependent upon coordinated regulation of gene expression by key regulators: transcription factors and mikroRNAs. PU.1 (Sfpi1) is a versatile hematopoetic transcription factor required for the proper generation of both myeloid and lymphoid lineages. MikroRNAs represent a novel class of ~22 nucleotide long non-coding posttranscriptional regulators that inhibit expression of genes by blocking protein translation or by mRNA degradation. In this PhD thesis I present research data documenting novel mechanisms of regulation and function of two oncogenic mikroRNAs, miR-17-92 cluster and miR-155 and myeloid transcriptional factors PU.1 upon macrophage differentiation of myeloid progenitors. The miR-17-92 cluster (Oncomir1) encodes seven related mikroRNAs that regulate cell proliferation, apoptosis and development and is overexpressed in number of malignancies including myeloid leukemia. Presented PhD thesis documents novel macrophage specific regulatory mechanisms involving the oncogenic cluster miR-17-92. Using transgenic PU.1-/- myeloid progenitors we show that upon macrophage differentiation, the transcription factor PU.1 induces the secondary determinant, the transcription...
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