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Noncanonical human eIF4Es in and out of the RNA granules
Frydrýšková, Klára ; Pospíšek, Martin (advisor) ; Půta, František (referee) ; Valášek, Leoš (referee)
Eukaryotic translation initiation factor eIF4E1 (eIF4E1) plays a pivotal role in the control of cap-dependent translation initiation, occurs in P- bodies and is important for the formation of stress granules (SG). Human cells encompass two other non-canonical translation initiation factors capable of cap binding although with a lower affinity for the cap: eIF4E2 and eIF4E3. Here, I investigated the ability of individual eIF4E family members and their variants to localize to SGs and P-bodies in stress-free, arsenite and heat shock conditions. Under all tested conditions, both eIF4E1 and eIF4E2 proteins and all their variants localized to P-bodies unlike eIF4E3 protein variants. Under both arsenite and heat stress conditions all tested variants of eIF4E1 and the variant eIF4E3-A localized to SGs albeit with different abilities. Protein eIF4E2 and all its investigated variants localized specifically to a major part of heat stress-induced stress granules. Further analysis showed that approximately 75% of heat stress-induced stress granules contain all three eIF4Es, while in 25% of them eIF4E2 is missing. Large ribosomal subunit protein L22 was found specifically enriched in arsenite induced SGs. Heat stress-induced re- localization of several proteins typical for P-bodies such as eIF4E2, DCP-1, AGO-2...
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The characteristics of stress granules in yeast Saccharomyces cerevisiae
Slabá, Renata ; Hašek, Jiří (advisor) ; Binarová, Pavla (referee)
9 ABSTRACT For proper function proteins should have a native conformation. If their conformation is impaired due to environmental stress or genetic mutation, proteins become prone to aggregation. There exist various types of protein aggregates. Stable non-membraneous inclusions can form which can serve for clearance of aberrant proteins from place where they can interfere with essential cellular processes. Another type of aggregates can serve as transient deposits of proteins thus protecting them from stress conditions. Stress granules (SG) are a such example of transient granules. Their formation is induced by heat shock for example. SGs contain mRNA, components of translation machinery, and other proteins. One of these proteins is Mmi1, small highly conserved protein with unknown function. Association of Mmi1 with stress granules and partial co-localization with chaperon Cdc48 and proteasom indicates Mmi1 can mediate heat stress damaged protein degradation. We have uncovered that yeast prion protein Sup35 is a component of stress granules as well. With regard to its aggregation capability there existed an assumption that prion domain of Sup35 could serve as scaffold for SG assembly. However as we show deletion of prion domain of Sup35 protein does not affect stress granules formation dynamics. Yeast...
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Human 4E protein family in stress granules granules and their further characterization
Hrbková, Pavlína ; Frydrýšková, Klára (advisor) ; Hašek, Jiří (referee)
Eukaryotic initiation factor 4E (eIF4E) is a key part of initiation and regulation of translation in human cells. Three members of human eIF4E proteins have been characterized: eIF4E1, eIF4E2 and eIF4E3. Cellular stress causes translation initiation inhibition followed by disassembly of the polysomes, those processes are accompanied by the assembly of cytoplasmic RNA granules, called stress granules (SG). Stress granules are dynamic structures whose composition may vary depending on the cell type and the stress stimulus. In this study, human cells were subjected to the following stress conditions: high temperature (HS), sodium arsenite (AS) or hypoxia. Using fluorescence microscopy, pairs of human translational initiation factors from the 4E protein family were visualized and their localization to SG was assessed with one GFP- 4E incorporated in the stable cell line and the other one detected endogenously. Here we show eIF4E1 being a part of all the SGs, both in HS and AS conditions. Next, the eIF4E1 and eIF4E3 proteins together form more SGs than proteins eIF4E1, respectively eIF4E3, with eIF4E2. And last, that the presence of the particular 4E protein has no effect on the composition of SGs. Furthermore, selected groups of proteins were assessed for their potential to localize to the SGs under HS...
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Formation of protein granules in differentiated cells of yeast colonies
Kočířová, Eliška ; Palková, Zdena (advisor) ; Heidingsfeld, Olga (referee)
Saccharomyces cerevisiae is a unicellular eukaryotic organism capable of forming organized multicellular communities - colonies and biofilms. During development, colonies of laboratory strains differentiate into specifically localized cell subpopulations - U and L cells, located in the upper and lower part of the colony, respectively. The U and L subpopulations of cells vary in morphology, metabolic processes and stress resistance. Protein granules are membrane-less "organelles" found in both unicellular and multicellular eukaryotic organisms. The formation of protein granules is related to the physiological state of the cell (e.g. chronological and replicative aging), but also to changing environmental conditions and to cellular responses to stress factors. A relatively large fraction of proteins relocalizes to some type of protein granule during the lifespan of the cell. Granule formation can increase fitness of cells, help them to cope with limiting energy resources, and plays a crucial role in the adaptation of cells to stress conditions. Localization of many proteins in the cell varies depending on its physiology. Therefore the specific localization of such proteins may be considered as a "marker" of a specific physiological condition. There are proteins in each type of granule that can be...
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The role of stress granules and 4E-BP in heat-stressed cells of S. cerevisiae
Kolářová, Věra ; Hašek, Jiří (advisor) ; Zimmermannová, Olga (referee)
The cells are capable of very quick and specific reactions on stress conditions. Influence of translation, specifically initiation of translation by inhibition factors, is one of the main regulatory process. Two of eIF4E-binding proteins (4E-BP), Eap1p and Caf20p, are known as cap-dependent translation repressors in yeast Saccharomyces cerevisiae. We used in vivo fluorescent microscopy analysis to show different reaction of Caf20p and Eap1p to heat stress. Protein Caf20p does not react on heat shock and stays difused in cytoplasm. Contrary to Caf20p reaction, protein Eap1p accumulates in cytoplasm close to stress granules (SGs). This work shows that Eap1p is involved in stress granules assembly. In the absence of Eap1p, yeast cells react to the heat stress with small and less focused SGs. Dele- tion of CAF20 does not affect SG assembly. This points to specific function of SG in distribution of factors connected with stress reaction. Polysomal analysis shows that deletion of one of initiation translation repressors does not affect heat induced global repression of translation. In permisive condition deletion of EAP1 may cause defect in addition of 60S ribosomal subunits. Absence of protein Eap1p causes morphological defect. That point to a different reactions of Eap1p and Caf20p on heat stress and possible...
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Dynamics of ALBA proteins in Arabidopsis thaliana evaluated by fluorescence microscopy
Popelářová, Anna ; Honys, David (advisor) ; Vosolsobě, Stanislav (referee)
ALBA proteins were discovered in Archaea more than 30 years ago. They were gradually identified to be well conserved in Eucaryotes as well. A functional dimeric form of these proteins with DNA and RNA-binding capability was claimed in both mentioned domains of organisms. However, their roles diversified during evolution and vary in between organisms. In Archaea, ALBAs are involved in the genome organization and RNA-protein interactions. In Eukaryotes, there are presented two different subfamilies of ALBA proteins - Rpp20 and Rpp25 subfamily. A sole protein from each subfamily was identified in some organisms though they were multiplied in plants, respectively. These proteins can interact with each other and participate in ontogenetic development and stress responses. According to several studies, ALBA proteins were found to be involved in DNA stability maintenance or pre-rRNA splicing in the nucleus of Arabidopsis thaliana. However, they have been shown to play a role in the cellular metabolism and stress responses in cytoplasm. Six ALBA proteins were identified in the genome of A. thaliana, three from each subfamily. In this study, all heterodimeric protein- protein interactions were investigated by the bimolecular fluorescence complementation (BiFC) assay which revealed positive results in...
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Formation of protein granules in differentiated cells of yeast colonies
Kočířová, Eliška ; Palková, Zdena (advisor) ; Heidingsfeld, Olga (referee)
Saccharomyces cerevisiae is a unicellular eukaryotic organism capable of forming organized multicellular communities - colonies and biofilms. During development, colonies of laboratory strains differentiate into specifically localized cell subpopulations - U and L cells, located in the upper and lower part of the colony, respectively. The U and L subpopulations of cells vary in morphology, metabolic processes and stress resistance. Protein granules are membrane-less "organelles" found in both unicellular and multicellular eukaryotic organisms. The formation of protein granules is related to the physiological state of the cell (e.g. chronological and replicative aging), but also to changing environmental conditions and to cellular responses to stress factors. A relatively large fraction of proteins relocalizes to some type of protein granule during the lifespan of the cell. Granule formation can increase fitness of cells, help them to cope with limiting energy resources, and plays a crucial role in the adaptation of cells to stress conditions. Localization of many proteins in the cell varies depending on its physiology. Therefore the specific localization of such proteins may be considered as a "marker" of a specific physiological condition. There are proteins in each type of granule that can be...
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Role of RACK1 in translation regulation during stress conditions
Chvalová, Věra ; Groušl, Tomáš (advisor) ; Převorovský, Martin (referee)
RACK1 (Receptor for activated C kinase 1) is an evolutionary conserved protein which has essential role in most studied eukaryotic organisms, except for yeast. Although RACK1 was originally described as a binding partner of protein kinase C, later studies re- vealed its significant role in other cellular signalizations such as MAPK, Src or FAK. Thanks to this, RACK1 participates in the regulation of key cellular processes including migration, apoptosis or translation. As a binding partner of a small ribosomal subunit, RACK1 contributes to transla- tion regulation by integrating signals from different cellular pathways and several transla- tional components such as PKC and eIF6. Moreover, RACK1 has a role in translation regu- lation during stress. Under stress conditions there is a global reduction of translation, in- creased expression of specific mRNAs important for cellular stress response and formation of cytosolic foci called stress granules (SGs). SGs play an important role in protection of mRNAs and translation components against degradation. SGs also function in prevention of apoptosis. RACK1 has been identified as one of many components of SGs and its localization into SGs leads to inhibition of RACK1-mediated pro-apoptotic pathways. Aim of this diploma thesis was to elucidate the role of...
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Noncanonical human eIF4Es in and out of the RNA granules
Frydrýšková, Klára ; Pospíšek, Martin (advisor) ; Půta, František (referee) ; Valášek, Leoš (referee)
Eukaryotic translation initiation factor eIF4E1 (eIF4E1) plays a pivotal role in the control of cap-dependent translation initiation, occurs in P- bodies and is important for the formation of stress granules (SG). Human cells encompass two other non-canonical translation initiation factors capable of cap binding although with a lower affinity for the cap: eIF4E2 and eIF4E3. Here, I investigated the ability of individual eIF4E family members and their variants to localize to SGs and P-bodies in stress-free, arsenite and heat shock conditions. Under all tested conditions, both eIF4E1 and eIF4E2 proteins and all their variants localized to P-bodies unlike eIF4E3 protein variants. Under both arsenite and heat stress conditions all tested variants of eIF4E1 and the variant eIF4E3-A localized to SGs albeit with different abilities. Protein eIF4E2 and all its investigated variants localized specifically to a major part of heat stress-induced stress granules. Further analysis showed that approximately 75% of heat stress-induced stress granules contain all three eIF4Es, while in 25% of them eIF4E2 is missing. Large ribosomal subunit protein L22 was found specifically enriched in arsenite induced SGs. Heat stress-induced re- localization of several proteins typical for P-bodies such as eIF4E2, DCP-1, AGO-2...
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Human 4E protein family in stress granules granules and their further characterization
Hrbková, Pavlína ; Frydrýšková, Klára (advisor) ; Hašek, Jiří (referee)
Eukaryotic initiation factor 4E (eIF4E) is a key part of initiation and regulation of translation in human cells. Three members of human eIF4E proteins have been characterized: eIF4E1, eIF4E2 and eIF4E3. Cellular stress causes translation initiation inhibition followed by disassembly of the polysomes, those processes are accompanied by the assembly of cytoplasmic RNA granules, called stress granules (SG). Stress granules are dynamic structures whose composition may vary depending on the cell type and the stress stimulus. In this study, human cells were subjected to the following stress conditions: high temperature (HS), sodium arsenite (AS) or hypoxia. Using fluorescence microscopy, pairs of human translational initiation factors from the 4E protein family were visualized and their localization to SG was assessed with one GFP- 4E incorporated in the stable cell line and the other one detected endogenously. Here we show eIF4E1 being a part of all the SGs, both in HS and AS conditions. Next, the eIF4E1 and eIF4E3 proteins together form more SGs than proteins eIF4E1, respectively eIF4E3, with eIF4E2. And last, that the presence of the particular 4E protein has no effect on the composition of SGs. Furthermore, selected groups of proteins were assessed for their potential to localize to the SGs under HS...
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