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Interplay between the tRNA anticodon stem and small ribosomal proteins forming the decoding site during stop codon readthrough
Čapková, Zuzana ; Valášek, Leoš (advisor) ; Eliáš, Marek (referee) ; Macíčková Cahová, Hana (referee)
Transfer RNAs (tRNAs) represent an indispensable part of protein synthesis by delivering amino acids into ribosomal A site during elongation. The rules that govern the correct match between the tRNA anticodon and the A-site mRNA codon are set by the genetic code. Specifically, the code defines that 61 sense codons are recognized by tRNAs and 3 stop codons marking translation termination are recognized by the release factor. In certain circumstances, a near-cognate (or a cognate) tRNA can be incorporated at the stop codon and translation elongation proceeds further. This process, called stop codon readthrough, may be beneficial as a therapy for inherited genetic diseases caused by premature termination codons (PTCs), and importantly, it is crucial for organisms with stop codon reassignment(s). Here I show that apart from the anticodon itself, the peculiar anticodon stem of two different near-cognate tRNAs, namely yeast S. cerevisiae tRNAGln CUG and Blastocrithidia nonstop tRNATrp CCA, is critical for their readthrough promoting potential. In particular, yeast tRNAGln CUG relies on the specific pyrimidine 28 : purine 42 base pair forming the 4th pair of its anticodon stem, whereas tRNATrp CCA of B. nonstop acquired a mutation that shortened its anticodon stem from the canonical 5 to the 4 base pairs,...
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tRNA synthetases as potential RNA capping enzymes
Říha, Jan ; Macíčková Cahová, Hana (advisor) ; Brázdovič, Filip (referee)
(EN) A novel type of non-canonical 5′ RNA caps, dinucleoside polyphosphate RNA caps, has recently been discovered in our laboratory. To elucidate the physiological role of these caps, more detailed information about their synthesis is essential. It has been already described that dinucleoside polyphosphate RNA caps (NpnN-RNA caps) can be accepted by RNA polymerase during transcription as a non-canonical initiating nucleotide (NCIN). However, the possibility that NpnN-RNA caps are created post- transcriptionally cannot be ruled out. The best candidates for post-transcriptional capping enzymes are aminoacyl-tRNA synthetases, which, besides their crucial role in translation, are responsible for the synthesis of free dinucleoside polyphosphates. In this thesis, four E. coli tRNA synthetases have been selected, cloned into plasmids, and purified using fast protein liquid chromatography (FPLC). Subsequently, selected tRNA synthetases have been tested for the production of free dinucleoside polyphosphate. These experiments have identified the optimal conditions for production of free dinucleoside polyphosphates, diadenosine tetraphosphate (Ap4A) particularly. The tRNA synthetases were then tested for their capabilities to form an RNA cap on in vitro transcribed radioactively labelled RNA. We found that...
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Methylation of viral RNA
Šimonová, Anna ; Macíčková Cahová, Hana (advisor) ; Sýkora, David (referee) ; Elleder, Daniel (referee)
Viruses are the major force that shapes the evolution of both pro- and eukaryotic organisms. They have a simple inner organization and contain only a few, usually well-described RNAs. In the case of +(ss)RNA viruses, their genomic RNA serves also as mRNA. This makes them a perfect model system for searching for new mRNA modifications as well as for understanding the role of already known modifications. In this work, Human Immunodeficiency Virus type 1 (HIV-1) from the Retroviridae family was used as a model system. In the following study, four representatives from the Picornaviridae family were tested for RNA methylation profile. To get the information, a combination of two techniques was developed, liquid chromatography- mass spectrometry (LC-MS) and sequencing techniques. Results of LC-MS reveal a surprisingly high amount of 1-methyladenosine (m1 A) in RNA isolated from HIV-1. Nevertheless, the m1 A mapping sequencing technique confirm m1 A position only in co-packed tRNA. This led to the recalculation of HIV-1 virion RNA composition. In the case of Picornaviridae, LC-MS revealed m1 A and 5-methylcytidine (m5 C) in two insect viruses (Sacbrood virus, SBV and Deformed wing virus, DWV). RNA seq techniques (m1 A mapping and bisulfite sequencing) confirmed the presence of m1 A and m5 C only in tRNA....
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Intracellular dinucleoside polyphosphates and methods of their detection
Říha, Jan ; Macíčková Cahová, Hana (advisor) ; Šanderová, Hana (referee)
(EN) Dinucleoside polyphosphates are already known for more than 50 years, but their role inside a cell is still unclear. Some theories discuss their possible role as alarmones during stress conditions, others connect them to DNA damage or proliferation. One new theory is that dinucleoside polyphosphates act as 5' RNA caps. To elucidate their role in organisms, it is important to know their concentration in normal and stress conditions. This work will try to determine basal concentration in both bacterial and eukaryotic cells, and the changes of their concentration under stress conditions, from already known data. Measurement of concentration of any compound inside a cell depends on the used method. I also present basic overview of methods for detecting dinucleoside polyphosphates, from older luciferase- based techniques to new precise mass spectrometry-based techniques. Keywords: dinucleoside polyphosphates, Ap4A, RNA caps, cellular stress, LC-MS detection and quantification
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Ap4A-RNA in IgE activated mast cells
Potužník, Jiří František ; Macíčková Cahová, Hana (advisor) ; Černý, Jan (referee)
Mast cells are tissue resident members of the immune system. They have a wide range of functions and receptors including the FcεRI receptor, which gets activated by binding to IgE bound to an antigen. When the cells are activated in this manner, a process termed the LysRS- Ap4A-MITF signalling pathway occurs, resulting in the translocation of the Lys tRNA synthetase into the nucleus and an activation of its moonlighting activity - the production of diadenosine tetraphosphate (Ap4A). Ap4A is a dinucleoside polyphosphate, a type of ubiquitous molecule present in all domains of life. They are made up of two nucleosides joined together by a 5' to 5' phosphodiester bridge of variable lengths. Recently, these molecules have been shown to serve as non-canonical initiating nucleotides during bacterial transcription, where they function as 5' RNA caps, similar to the well-known 7- methylguanosine eukaryotic mRNA cap. In this thesis, I present proof of existence of Ap 4A capped RNA in mast cells, a previously unknown 5' RNA structure in eukaryotic cells, and I attempt to pinpoint its role in the activation of these cells and in the wider context of mast cell mediated immune response. Keywords: mast cells, RNA caps, Dinucleoside polyphosphates, Ap 4A, RNA modification, IgE, FcεRI receptor, Lysine tRNA synthetase
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Characterization of non-canonical RNA polymerase encoded by the yeast linear plasmids
Sýkora, Michal ; Vopálenský, Václav (advisor) ; Macíčková Cahová, Hana (referee) ; Valášek, Leoš (referee)
Transcription is the control point of gene expression. This process relies on protein complex of multisubunit RNA polymerases, which are extremely conserved among all cellular organisms. Transciption of extrachromosomal hereditary elements such as organelles, viruses and plasmids is dependent on host cellular RNA polymerases or intrinsic RNA polymerase is contained within these elements. Putative non-canonical two-subunit RNA polymerase is also encoded by linear cytoplasmic plasmids of the yeast Kluyveromyces lactis and most likely transcribes genes of these plasmids. Besides the two subunits of RNA polymerase encoded by linear plasmids of Kluyveromyces lactis there are another two estimated components of the transcription apparatus, namely capping enzyme that adds the cap to 5' mRNA ends and putative DExD/H box helicase. Characterization of the unique and underexplored transcription machinery of Kluyveromyces lactis plasmids was the principal objective of this work. The main goal was to: 1) clarify evolutionary origin of the linear plasmid transcription apparatus; 2) describe architecture of the linear plasmid transcription complex in vivo focused on putative RNA polymerase binding partners; 3) reveal mechanisms of transcription initiation and termination of the yeast linear plasmids. The main...
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The function of 2'-O-methylated RNA in the context of viral infection
Potužník, Jiří ; Macíčková Cahová, Hana (advisor) ; Forstová, Jitka (referee)
RNA is subject to a wide array of post-transcriptional modifications. 2'-O-methylation is an essential intrinsic modification of RNA. It affects the structure and reactivity of the molecule as well as its function. 2'-O-methylation is highly conserved, present in all three domains of life. Viral RNA uses this modification to mimic the host and evade detection by the immune system. There are two main mechanisms, through which viral 2'-O-methylated RNA does this. The first is evading detection by a pattern recognition receptor form the RIG-I-like receptor family Mda5. Mda5 is capable of detecting unmethylated RNA and recognising it as non-self, thus initiating an immune response. The second mechanism the evasion and restriction of an effector molecule IFIT. IFIT proteins are capable of detecting the absence of 2'-O- methylation on viral RNAs and inhibiting their translation. They do this by interfering with the formation of the ternary complex, an essential member of ribosomal formation. Using viral 2'- O-methylation as a target for therapy, it is possible to develop attenuated vaccines. Keywords: viral RNA, RNA modifications, 2'-O-methylation, Mda5, IFIT, RIG-I-like receptors, epitranscriptomics, WNV, JEV
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Magnetic Beads-Based Electrochemical Techniques for DNA-Protein Interaction Monitoring
Fojta, Miroslav ; Pivoňková, Hana ; Němcová, Kateřina ; Horáková Brázdilová, Petra ; Havran, Luděk ; Orság, Petr ; Vidláková, Pavlína ; Macíčková-Cahová, Hana ; Balintová, Jana ; Hocek, Michal
Electrochemical techniques, in connection with separation of nucleoprotein complexes at magnetic beads, are suitable for the monitoring of DNA-protein interactions. For the detection of complexes captured at the beads it is possible to utilize intrinsic electrochemical activity of the protein, intrinsic structure-selective signals of the DNA, or indicator DNA substrates tail-labeled with electroactive moieties.
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Electrochemical analysis of DNA using switchable redox moieties
Fojta, Miroslav ; Daňhel, Aleš ; Horáková Brázdilová, Petra ; Plucnara, Medard ; Pivoňková, Hana ; Havran, Luděk ; Vidláková, Pavlína ; Raindlová, Veronika ; Balintová, Jana ; Macíčková-Cahová, Hana ; Hocek, Michal
Labelling of DNA with electrochemically active moieties proved to be a convenient way to the development of electrochemical techniques for the sequence-specific DNA sensing. Through combinations of various labels differing in redox potentials, independent redox coding of different DNA sequences or individual nucleobases can be attained. Applications possibilities of electrochemistry in analysis of modified DNAs are further extended by facile monitoring of chemical conversion of reactive groups on DNA during post-labelling with ultimate redox labels. In addition, controlled in situ electrochemical conversions of specific intrinsic and extrinsic DNA components can be utilized to switch their electrochemical signals and improve signal resolution.
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