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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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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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Induction of endogenous RNAi in mammalian cells
Demeter, Tomáš ; Svoboda, Petr (advisor) ; Vopálenský, Václav (referee)
Double-stranded RNA (dsRNA), a double helix formed by two antiparallel complementary RNA strands, is a unique structure with a variety of biological effects. dsRNA can be introduced into the cell from exogenous sources or it can be produced endogenously. There are four basic mechanisms producing dsRNA: inverted repeat transcription, convergent transcription, pairing of sense and antisense RNAs produced in trans, and RNA dependent RNA polymerase-mediated synthesis dsRNA. Different mechanisms of production determine additional structural features of dsRNA, such as dsRNA termini, mismatches etc. These features may affect cellular response to dsRNA. Recognition of dsRNA can trigger several responses that act in sequence-specific or sequence-independent manners. The main sequence- specific response triggered by dsRNA is RNA interference (RNAi) is. Our laboratory has been studying mechanism of induction of RNAi in mammalian cells using one specific type of long dsRNA expression system. The dsRNA used in these experiments formed hairpin structure with long 5' and 3' single-strand RNA overhangs. We hypothesized that other dsRNA substrates might be more efficient than the one used in mammalian RNAi experiments since 2002. Accordingly, the main aim of my thesis was to compare efficiency of different dsRNA...
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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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Analysis of short Argonaute isoforms from mouse oocytes
Jankele, Radek ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee)
AnalysisofshortArgonauteisoformsfrommouseoocytes Abstract: Argonaute proteins carrying small RNAs form the conserved core of RNA silencing mechanisms, which repress viruses, mobile genetic elements, and genes in a sequence specific manner. The microRNA (miRNA) pathway is a dominant mammalian RNA silencing mechanism in somatic cells, which post-transcriptionally regulates large fraction of genes and thereby adjusts protein levels. miRNA-guided Argonautes inhibit translation and induce deadenylation of complementary mRNAs, ultimately resulting in their decay. In contrast to RNA interference (RNAi), which employs Argonaute slicer activity to directly cleave perfectly complementary RNAs, an effective miRNA-mediated mRNA repression requires multiple Argonaute-associated protein factors and enzymes. The miRNA pathway has been implicated in many complex biological processes ranging from organogenesis, stress-response to haematopoiesis or cancer. Surprisingly, canonical miRNAs are not essential for oocytes and early embryonic development in mice. Even the most abundant miRNAs present in mouse oocytes are unable to effectively repress target genes. However, RNAi, which shares key enzymes with the miRNA pathway, is highly active in oocytes and early embryos. The cause of miRNA inactivity in mouse oocytes remains...
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