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Screening for the HCV IRES interacting proteins
Roučová, Kristina ; Pospíšek, Martin (advisor) ; Kuthan, Martin (referee)
Hepatitis C virus (HCV) is a worldwide spread pathogen infecting up to 3 % of the human population. Nowadays, research of new drugs against this virus is focused on the individual steps in its life cycle, including the translation initiation. In the case of HCV translation initiation is dependent on the internal ribosome entry site (IRES). Besides of components of the translational machinery also other components of the cell, so called IRES trans-acting factors (ITAF), contribute to its proper progress. This work continues in previous research of our laboratory focused on searching for new ITAF. In order to search for potential ITAF increasing HCV IRES activity new recombinant plasmid vectors and reference strains were prepared and selection conditions of the selection system were optimized. The differences in the growth characteristics of the reference strains were analyzed and quantified under selective and non-selective conditions. A set of pilot high efficiency transformations of the yeast strain pJ69-4A carrying bicistronic construct with HCV IRES were conducted using human expression cDNA library in order to optimize the efficiency of transformation and selection conditions and to attempt to identify new ITAF. Several dozens of randomly selected clones from these transformations obtained under...
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RNAi of the a subunit of human translation initiation factor 3 (eIF3).
Peclinovská, Lucie ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
Translation initiation is the first step of protein synthesis that captures the flow of gene expression pathway in all living organisms. The advantage of regulation of gene expression at the level of translation initiation is that it allows for more rapid changes in the proteome and serves as the rate limiting step under certain conditions such as stress. This process is masterminded by many initiation factors. One of them, a multisubunit eukaryotic initiation factor 3 (eIF3), is a very efficient player in this field taking a part in the most of the initiation steps. The largest subunit of the eIF3 complex is called eIF3a p170 and TIF32 in mammals and yeast, respectively, and at least in yeast, it was shown to represent an essential constituent of the translational machinery. This work is based on all that has been learned about the eIF3a roles in translation initiation in the model organism of yeast Saccharomyces cerevisiae in effort to examine the degree of the functional conservation with its human ortholog. This is achieved by the RNAi-mediated knock-down of eIF3a in HeLa and HEK cell lines followed by variety of well established assays to monitor translational status of eIF3a depleted cells. In the first part, I describe optimization of the RNA interference protocol with respect to the choice...
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Novel hepatitis C virus proteins
Zeman, Jakub ; Vopálenský, Václav (advisor) ; Horníková, Lenka (referee)
The hepatitis C virus (HCV) is a major etiological agent of chronic liver diseases. More than 170 million people worldwide are chronically infected, and more than 100 thousand of them develop hepatocellular carcinoma a year. HCV is an enveloped, positive-sense single-stranded RNA virus (+ssRNA virus) of the family Flaviviridae. Its genome is translated to produce a single polyprotein precursor that is further processed by cellular and viral proteases to form 10 viral proteins. Moreover, there is another protein encoded in an alternative reading frame. Two alternative translation mechanisms have been proposed for expression of this alternative reading frame protein (ARFP): a frameshift mechanism and translation initiating from internal start codons. Despite ten years of research its role in vivo is not yet explained. It appears that secondary structures in the core encoding region of HCV genome but not ARFP expression are required for robust viral translation and replication. The results of recent studies suggest that mutations distorting these structures may result not only in slowing down the viral cycle but also in a brand new and utterly unusual serological profile in patients as well as an increased level of expression of ARFP.
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mTORC1 complex function in regulation of translation initiation
Holásková, Lucie ; Feketová, Zuzana (advisor) ; Čáp, Michal (referee)
My bachelor thesis deals with the effect of mTOR pathway to different processes in the cell. In particular, it focuses on the influence of translation initiation. mTOR protein is part of two complexes, which occur in different organisms - mTORC1 and mTORC2. Eukaryotic initiation factor 4E (eIF4E) plays an important role in controlling translation initiation. The activity of eIF4E protein is regulated by family of repressor 4E-binding proteins (4E-BPs). Linking these proteins to eIF4E is regulated by their phosphorylation state. For the release of 4E-BP1 from eIF4E, phosphorylation must occur at four phosphorylation sites (Thr37, Thr46, Ser65 and Thr70). The study also covers some of the other events that occur in the mTOR pathway.
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The role of eIF3 in mRNA recruitment to the 43S pre-initiation complex
Beznosková, Petra ; Mašek, Tomáš (referee) ; Valášek, Leoš (advisor)
Translation initiation in eukaryotes plays an important role in gene expression and relies on interactions between many eukaryotic initiation factors, small and large ribosomal subunits, mRNA and initiator tRNA. The largest of these initiation factors, the eukaryotic initiation factor 3 (eIF3), participates in the most of translation initiation reactions. In yeast, eIF3 occurs together with eIF1, eIF5 and ternary complex (TC, eIF2-GTP-Met-tRNAi Met complex) in the multifactor complex (MFC) and promotes formation of the 43S preinitiation complexes. eIF3 also stimulates mRNA recruitment to the 43S preinitiation complexes, scanning the 5' untranslated region of mRNA for AUG recognition and has an important role in the gene-specific translational control mechanism called reinitiation involving short upstream open reading frames (uORFs).
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