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Identification of RNA elements in fungi
PÍCHALOVÁ, Barbora
Nowadays, the focus of fungus, mycovirus and environmental interactions has expanded. The kingdom of Fungi includes ecologically and economically important species of fungi that are pathogenic or symbiotic or can occur in the soil as mycorrhizal and endophytic fungi. Despite their ecological significance, biomedical and industrial importance, phylogenomic studies of Fungi and their viruses are lacking. To understand the basic interactions between mycoviruses and their fungal hosts, the study of the biological properties, distribution and transmission is required. This study aims to identify different species of fungi and determine the interaction between fungus and virus. Samples collected in the Czech Republic during 2020 and 2021 were used for diversity research and molecular genetic methods were chosen for their detection and identification. DNA was isolated using CTAB-PVP, the DNA segment was amplified and phylogenetically analyzed using the sequences of internal transcribed spacers (ITS), the translation elongation factor (TEF) 1-alpha gene and the beta-tubulin gene. Based on the sequences, a phylogenetic tree was created for the samples according to the genetic similarity between the individual genera. The method of dsRNA isolation using phenol-chloroform and cellulose was used for the detection of mycoviruses.
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Recognition of expressed double-stranded RNAs in mammalian cells
Vaškovičová, Michaela ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee)
Long double-stranded RNA (dsRNA) is a unique structure formed during viral replication or transcription of repetitive elements. Mammalian cells evolved several mechanisms how to respond to dsRNA. dsRNA can be engaged in one of three pathways: interferon response, RNA editing, and RNA interference (RNAi). RNAi is evolutionary conserved effect of dsRNA, which results in sequence-specific messenger RNA degradation. However, in mammals, RNAi is functional only in mouse oocytes, which express truncated version of Dicer (DicerO ). In somatic cells, dsRNA triggers sequence-independent interferon pathway. The main aim of this Master's thesis was to examine how specific double-stranded RNA-binding proteins (DRBPs) influence distribution of long dsRNA into RNAi and sequence-independent pathways. We used a luciferase-based reporter RNAi assay to monitor sequence-specific and sequence-independent effects of dsRNA co-expressed with selected DRBPs. Our results suggest that none of the tested DRBPs is sufficient to stimulate RNAi in somatic cells. Interestingly, the overexpression of either TARBP2 or PACT suppressed RNAi in cells expressing DicerO . Moreover, microRNA pathway, which employs the same protein factors as RNAi, is not inhibited by TARBP2 or PACT. Therefore, we propose that DRBPs overexpression...
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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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RNA interference in mouse oocytes and somatic cells
Táborská, Eliška ; Svoboda, Petr (advisor) ; O´Connell, Mary Anne (referee) ; Petr, Jaroslav (referee)
RNA interference (RNAi) is a pathway, which employs Dicer to process long double stranded RNAs (dsRNA) from endogenous or exogenous sources into short interfering RNAs (siRNA). siRNAs are loaded onto Argonaute proteins to mediate sequence-specific post-transcriptional RNA targeting resulting in regulation of protein-coding genes and retrotransposons or antiviral immune response. Another small RNA pathway - PIWI-associated RNA (piRNA) pathway is suppressing retrotransposons in the germline. In mice, canonical RNAi pathway activity is negligible in somatic cells where a full-length Dicer produces gene-regulatory microRNAs (miRNA) but RNAi is highly active in oocytes, which express a truncated oocyte-specific Dicer isoform (DicerO ). DicerO lacks an N-terminal DExD helicase domain and has higher cleavage activity of long dsRNAs. Deletion of oocyte specific DicerO promoter leads to transcriptome aberrations, which include upregulation of putative RNAi targets and MT retrotransposons and, consequently, to meiotic spindle defects and female sterility. In contrast, the piRNA pathway is non-essential in mouse oocytes, potentially because of overlapping functions of RNAi. The PhD thesis aims to understand biological significance of mammalian endogenous RNAi and to explore consequences of re-activated RNAi...
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Comparison of methods of isolating double-stranded RNA from plant tissue with a focus on viral fraction yield.
MATYÁŠOVÁ, Alena
High-throughput sequencing is one of methods used for diagnostics of plant pathogens and has advantage of unspecific unbiased detection of all nucleic acids present in a sample. Input material for HTS can be prepared using by different approaches that reflect purpose of the planned task. During viral infections of plants, viral double stranded RNAs are generated as replication intermediates or transcription products. Thus, they are often used for HTS. Nevertheless, such preparations contain large amount of plant RNAs. The work aimed for comparison of two methods of double stranded RNA enrichment - widely used cellulose chromatography and differential centrifugation with lithium chloride. Both qualitative and quantitative profiles of viral nucleic acids were estimated. An isolate HZ2 of red clover (Trifolium pratense L.) was used for the study. Previously, there were detected eight different RNA viruses with HTS-aimed analyses in the plant. To compare qualitative profile, RNA was extracted by each method, transcribed into cDNA, and specific viral fragments were amplified using PCR, followed by agarose gel electrophoresis. Quantitative profiles were analyzed using three selected viruses with single- and double-stranded RNA genomes. Their relative quantification was estimated using RT-qPCR approach. Further, normalized (to 26S rRNA as a reference) expressions were calculated using Bio-rad CFX Manager 3.1 software. All eight viruses were successfully detected using RNA material obtained by each method. After evaluating the quantification data and performing statistical tests (F-test, T-test; with a significance level of = 0.05), no significant difference was found between the compared methods.
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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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Recognition of expressed double-stranded RNAs in mammalian cells
Vaškovičová, Michaela ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee)
Long double-stranded RNA (dsRNA) is a unique structure formed during viral replication or transcription of repetitive elements. Mammalian cells evolved several mechanisms how to respond to dsRNA. dsRNA can be engaged in one of three pathways: interferon response, RNA editing, and RNA interference (RNAi). RNAi is evolutionary conserved effect of dsRNA, which results in sequence-specific messenger RNA degradation. However, in mammals, RNAi is functional only in mouse oocytes, which express truncated version of Dicer (DicerO ). In somatic cells, dsRNA triggers sequence-independent interferon pathway. The main aim of this Master's thesis was to examine how specific double-stranded RNA-binding proteins (DRBPs) influence distribution of long dsRNA into RNAi and sequence-independent pathways. We used a luciferase-based reporter RNAi assay to monitor sequence-specific and sequence-independent effects of dsRNA co-expressed with selected DRBPs. Our results suggest that none of the tested DRBPs is sufficient to stimulate RNAi in somatic cells. Interestingly, the overexpression of either TARBP2 or PACT suppressed RNAi in cells expressing DicerO . Moreover, microRNA pathway, which employs the same protein factors as RNAi, is not inhibited by TARBP2 or PACT. Therefore, we propose that DRBPs overexpression...
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Studium diversity a rozšíření virů entomopatogenní houby \nl{}\kur{Beauveria bassiana} v České republice
VANĚČEK, Petr
Mycoviruses are viruses that infect and replicate in fungal cells, but unlike most known viruses of plants and animals, they exceptionally produce deleterious effects on their host. Nonetheless, the last discoveries showed that some mycoviruses can decrease the virulence of their phytopathogenic fungal hosts, making them very attractive for their possible use as biological control agents. Most mycoviruses have dsRNA genomes and are widespread in all major taxa of fungi. Beauveria bassiana is one of the most studied species of entomopathogenic fungi; it has a cosmopolitan distribution and is used as biocontroller against invertebrates in agriculture. In the present work, a collection of 137 isolates of B. bassiana obtained at different locations and from different habitats in the Czech Republic was analysed. These isolates were analysed for the presence of dsRNA elements indicative of viral infections. The results revealed a high prevalence of viral infections in Czech B. bassiana isolates, with 22.6% of the isolates containing dsRNA elements with viral characteristics. Obtained dsRNA electropherotypes showed that virus diversity in infected isolates was high and that mixed virus infections occurred among them. Based on the characteristics of the electrophoretic band patterns, it could be hypothesized that B. bassiana isolates collected in the Czech Republic could harbour members of the viral families Totiviridae, Partitiviridae, Chrysoviridae and Hypoviridae.
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Occurrence of putative dsRNA mycoviruses in Ash Dieback Causal Agent
Čermáková, Vendula
Thanks to environmental changes, globalization, long distance trade and plant transport, invasive organisms have become a major threat for world biodiversity and ecosystem services. Over the last 20 years, common European ash trees (Fraxinus excelsior L., Fraxinus angustifolia Vahl. etc.) have been subjected to heavy dieback and mortality because of the introduction and spread of the ascomycetous fungal pathogen Hymenoscyphus pseudoalbidus Queloz (syn. Chalara fraxinea Kowalski). Once the disease is established, its management is hardly possible. Therefore, one of the main objectives of European researchers is to find effective and respectful control methods, such as biological control. The discovery of viruses which reduce the virulence of the chestnut blight fungus Cryphonecria parasitica (Murr.) Barr., has intensively stimulated the research of fungal viruses as potential biological control agents (BCA). The occurrence of putative dsRNA particles in the decaying fungus H. pseudoalbidus was investigated as an important indicator of the mycoviruses' presence. In total, 106 samples of this pathogen were obtained from eight different European countries. According to the results, dsRNA segments were confirmed in 32.1 % of examined samples (two similarly sized at 2--2.5 kb and a third one of approximately 5 kb). Statistical results have revealed no significant relation between the presence of dsRNA and growth rate, colour or any other characteristic of the mycelium.
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