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A study of the HCV IRES variability: An experimental approach coupled with design of a large-scale mutation database
Khawaja, Anas Ahmad ; Pospíšek, Martin (advisor) ; Hirsch, Ivan (referee) ; Valášek, Leoš (referee)
Translation initiation in the hepatitis C virus (HCV) occurs through a cap- independent mechanism that involves an internal ribosome entry site (IRES) capable of interaction with and utilization of the eukaryotic translational machinery. We focused on the structural configuration of the different HCV-IRES domains and the impact of IRES primary sequence variations on secondary structure conservation and function. For this purpose we introduced into our laboratory, methods such as denaturing gradient and temperature gradient gel electrophoresis for screening the degree of heterogeneity and total amount of HCV-IRES variability accumulated in HCV infected patients over a period of time. The selected samples showed variable migration pattern of the HCV-IRES (from all the patients) visualized in DGGE and TGGE, were sequenced and evaluated for translation efficiency using flow cytometry. In some cases, we discovered that multiple mutations, even those scattered across different domains of HCV-IRES, led to restoration of the HCV-IRES translational activity, although the individual occurrences of these mutations were found to be deleterious. We propose that such observation may be attributed to probable long- range inter- and/or intra-domain functional interactions. We established a large-scale HCV-IRES...
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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 polymerase: The "meeting point" of regulatory networks
Wiedermannová, Jana ; Krásný, Libor (advisor) ; Pospíšek, Martin (referee) ; Valášek, Leoš (referee)
Bacterial RNA polymerase (RNAP) is a multisubunit complex essential for transcription of DNA into RNA. As a key enzyme responsible for regulation of gene expression it interprets regulatory signals from the cell and based on these cues RNAP adjusts transcription level of particular genes. This process is affected both by the regular subunits of RNAP as well as other transcription factors (TFs) directly or indirectly interacting with RNAP. The general focus of this Thesis was to extend the knowledge about the complex transcriptional regulatory networks and about the connections between individual pathways. The main specific topic and the main publication of the thesis are focused on the HelD protein, a novel binding partner of RNAP in Bacillus subtilis. We showed that HelD binds between the secondary channel of RNAP and alpha subunits of the core form of the enzyme. We proved that HelD stimulates transcription in an ATP dependent manner by enhancing transcriptional cycling and elongation. We revealed a new connection in the transcription regulatory machinery when we demonstrated that the stimulatory effect of HelD can be amplified by delta, a small subunit of RNAP specific for gram positive (G+) bacteria. Two other publications of the thesis are dealing with the delta subunit. We solved the 3D...
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Mapping the contact points between eukaryotic translation initiation factor eIF3 and the 40S ribosomal subunit.
Kouba, Tomáš ; Valášek, Leoš (advisor) ; Pospíšek, Martin (referee) ; Staněk, David (referee)
Translation initiation in eukaryotes is a multistep process requiring the orchestrated interaction of several eukaryotic initiation factors (eIFs) together with the small ribosomal subunit to locate the mRNA's translational start and to properly decode the genetic message that it carries. The largest of these factors, eIF3, forms the scaffold for other initiation factors to promote their spatially coordinated placement on the ribosomal surface. It is our long-standing pursuit to map the 40S-binding site of the yeast multisubunit eIF3 and here we present three new mutual interactions between these two macromolecules (i) The C-terminal region of the eIF3c/NIP1 subunit is comprised of the conserved bipartite PCI domain and we show that a short C-terminal truncation and two clustered mutations directly disturbing the PCI domain produce lethal or slow growth phenotypes and significantly reduce amounts of 40S-bound eIF3 in vivo. The extreme C-terminus directly interacts with small subunit ribosomal protein RACK1/ASC1, which is a part of the 40S head, and, consistently, deletion of ASC1 impairs eIF3 association with ribosomes. The PCI domain per se shows strong but unspecific binding to RNA, for the first time implicating this protein fold in protein-RNA interactions. We conclude that the c/NIP1...
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Charakterizace dvou nejmensich podjednotek eIF3 a jejich úloh v translaci.
Cuchalová, Lucie ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Pospíšek, Martin (referee)
Protein synthesis or mRNA translation is a complex and highly conserved process. Translation consists of initiation, elongation, termination, and ribosome recycling stages. Since most regulation occurs during initiation, its mechanism is being studied intensively to elucidate the molecular basis of every potential control point. The initiation factor eIF3, which in yeast consists of five essential core subunits (eIF3a/TIF32, b/PRT1, c/NIP1, g/TIF35, and i/TIF34) and one transiently associated, non-essential subunit (j/HCR1), is undisputedly one of the key promoters of initiation. In addition, it has also been implicated in playing a critical role during ribosomal recycling, reinitiation, signal transduction, NMD etc. We have focused on determining the molecular mechanism of the roles of eIF3 and its associated eIFs not only in translation initiation but also in termination and in reinitiation. This included the biochemical and genetic mapping of yeast eIF3 binding site on the small ribosomal subunit, among others. We showed that the interaction between the residues 200-400 of a/TIF32-NTD and flexible C-terminal tail RPS0A significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo, thus a/TIF32-NTD together with the recently published PCI (proteasome...
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Complex structural and functional analysis of individual subunits of yeast translation initiation factor 3.
Herrmannová, Anna ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Pospíšek, Martin (referee)
5 ABSTRACT Translation initiation in eukaryotes is a complex process promoted by numerous proteins or protein complexes called eukaryotic initiation factors (eIFs). The eukaryotic initiation factor 3 (eIF3) is a multisubunit complex that has been implicated in several steps of the translation initiation pathway. In yeast Saccharomyces cerevisiae, eIF3 is composed of five essential subunits (a/TIF32, b/PRT1, c/NIP1, g/TIF35, i/TIF34) and one nonessential subunit (j/HCR1). It is our long-term goal to understand how eIF3 promotes different stages of translation initiation and which subunits or their domains play a critical role in this process as well as to map the binding sites of eIF3 on 40S ribosomal subunit and to create a structural model of eIF3 complex. Here I present two structural studies showing interactions between the RNA recognition motif of eIF3b and a short peptide of eIF3j subunits of human eIF3 solved by NMR spectroscopy, and a crystal structure of the C-terminal fragment of yeast b/PRT1 in complex with the full length i/TIF34 subunit at 2.2 Å resolution. In the former study, me and my colleagues showed that the critical determinants mediating this eIF3b-eIF3j interaction are evolutionary conserved, since their mutations in yeast proteins reduced cellular growth rate, eliminated j/HCR1...
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Elucidating the interactions of interleukin-1alpha with components of the eukaryotic transcription machinery
Zámostná, Blanka ; Pospíšek, Martin (advisor) ; Černý, Jan (referee) ; Mělková, Zora (referee)
4 ABSTRACT Interleukin-1α (IL-1α) is a pleiotropic cytokine and a key mediator of host immune response. It is synthesised as a 31-kDa precursor, that is cleaved by the cysteine protease calpain into the 17-kDa mature IL-1α and the 16-kDa N- terminal peptide of IL-1α (IL-1αNTP). Although IL-1α can be secreted, act on target cells through the surface receptor IL-1RI and trigger the signal transduction pathway, increasing evidence points toward the involvement of IL-1α in certain nuclear processes. IL-1αNTP is highly conserved among higher eukaryotes and contains a nuclear localisation sequence; indeed, both the precursor and IL-1αNTP are found in the cell nucleus. Previously, a genetic interaction of IL-1α with nuclear histone acetyltransferase (HAT) complexes has been reported from mammalian cells and, interestingly, also from the heterologous yeast model. This thesis extends the research of the nuclear function of IL-1α and demonstrates that IL-1α physically associates with the HAT/Core module of yeast SAGA and ADA HAT complexes. Results of the HAT subunit gene knock-out experiments followed by a set of co-immunoprecipitations also suggest a novel model of the yeast SAGA complex assembly, in which ADA appears to represent only a partly functional HAT complex. In its natural milieu of mammalian cells, IL-1α...
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Influence of rRNA modifications on translation initiation in eukaryots
Kročová, Eliška ; Pospíšek, Martin (advisor) ; Kouba, Tomáš (referee)
Modifications of ribosomal RNA are present in every livivng organism. The function of rRNA modifications could be studied only when the process of modifications was described. Currently, scientists study not only individual modifications but also the importance of global level of modifications for maturation and function of ribosome. This thesis deals with the influence of 2'-O-methylation of citidine 1639 and adenosine 100 in 18S rRNA and uridine 2729 in 25S rRNA on initiation in yeast Saccharomyces cerevisiae with special attention of translation controlled by internal ribosome entry site (IRES). Strains with deletion in genes snR51, snR70 and duoble deletion in both genes were successfully created during my master study. Pilot experiments showed the importance of products of both genes in translation initiation.
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Preparation of yeast system for investigation of the human translation initiation
Holásková, Lucie ; Pospíšek, Martin (advisor) ; Cuchalová, Lucie (referee)
Protein synthesis is principally regulated at the initiation stage in which eIF4F complex plays an important role. The eIF4F complex contains three subunits - eIF4A, eIF4E and eIF4G. The eIF4E is cap binding protein, the eIF4A is RNA dependent helicase which unwinds secondary structures at mRNA and scaffolding eIF4G protein. The interaction with other translation initiation factors is important for protein synthesis. The goal of my thesis was to create a new Saccharomyces cerevisiae yeast strain with the human eIF4F factor. Firstly I replaced yeast eIF4E protein with human eIF4E protein. I used a cre/loxP recombination to prepare yeast strains with deleted genes eIF4GI (huΔ4G1) and eIF4GII (huΔ4G2). Characterization of the new yeast strains showed that the human eIF4E protein replaced yeast ortholog factor better than the eIF4E protein from yeast Candida albicans. First experiments showed putative role of the eIF4GII protein during the cell growth under the temperature and osmotic stress. Key words: translation initiation, eIF4E, eIF4G, Saccharomyces cerevisiae
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