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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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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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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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