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The effects of long double-stranded RNA expression in mammalian cells.
Nejepínská, Jana ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee) ; Štefl, Richard (referee)
Double stranded RNA (dsRNA) is a foreign molecule that arises in the cell either as a by-product of viral replication or it is produced by the intramolecular or intermolecular pairing of complementary RNAs, often originating from repetitive sequences. In mammals, dsRNA can enter one of three pathways: the sequence-specific RNA silencing, the sequence-independent interferon (IFN) response, or editing by adenosine deaminases. The main focus of my PhD project was to comprehensively analyze the effects of the expressed dsRNA in mammals in the context of the whole organism. To follow this aim, we generated a construct expressing dsRNA in a form of an mRNA containing a long perfect hairpin structure. Transgenic mice ubiquitously expressing dsRNA were viable and, in contrast to the previous studies, the IFN response was not activated. In somatic cells, dsRNA was poorly processed into small interfering RNAs, did not cause transcriptional silencing in trans, and underwent low adenosine deamination without the nuclear retention. Consistent results were obtained in human cells transiently transfected with a dsRNA-expressing plasmid. On the other hand, dsRNA expression caused robust RNA interference (RNAi) in oocytes. Thus, we show for the first time that expressed dsRNA, in contrast to many other forms of...
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Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes
Zeman, Jakub ; Valášek, Leoš (advisor) ; Štefl, Richard (referee) ; Man, Petr (referee)
In eukaryotic translation, eukaryotic initiation factors (eIFs) are at least as important as the ribosome itself. Some of these factors play different roles throughout the entire process to ensure proper assembly of the preinitiation complex on mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most important factor integrating signals from others and coordinating their functions on the ribosome is eIF3. In Saccharomyces cerevisiae, eIF3 is formed by five subunits. All these subunits contain structural motifs responsible for contact with ribosomal proteins and RNAs. In addition to these highly structured parts, the rest of eIF3 is unstructured and very flexible. Therefore, despite the recent progress thanks to the use of a cryo-electron microscopy, a precise structure and position of eIF3 on the 40S ribosomal subunit are still not known. Also, the presence of eIF3 on 80S during early elongation and its role in reinitiation and readthrough are not fully understood. In order to crack mysteries of yeast eIF3, we used x-ray crystallography, chemical cross- linking coupled to mass spectrometry, and various biochemical and genetic assays. We demonstrated that eIF3 is very compactly packed when free in solution. This...
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The effects of long double-stranded RNA expression in mammalian cells.
Nejepínská, Jana ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee) ; Štefl, Richard (referee)
Double stranded RNA (dsRNA) is a foreign molecule that arises in the cell either as a by-product of viral replication or it is produced by the intramolecular or intermolecular pairing of complementary RNAs, often originating from repetitive sequences. In mammals, dsRNA can enter one of three pathways: the sequence-specific RNA silencing, the sequence-independent interferon (IFN) response, or editing by adenosine deaminases. The main focus of my PhD project was to comprehensively analyze the effects of the expressed dsRNA in mammals in the context of the whole organism. To follow this aim, we generated a construct expressing dsRNA in a form of an mRNA containing a long perfect hairpin structure. Transgenic mice ubiquitously expressing dsRNA were viable and, in contrast to the previous studies, the IFN response was not activated. In somatic cells, dsRNA was poorly processed into small interfering RNAs, did not cause transcriptional silencing in trans, and underwent low adenosine deamination without the nuclear retention. Consistent results were obtained in human cells transiently transfected with a dsRNA-expressing plasmid. On the other hand, dsRNA expression caused robust RNA interference (RNAi) in oocytes. Thus, we show for the first time that expressed dsRNA, in contrast to many other forms of...
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