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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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Structure and function of IRES elements in yeast
Černý, Jiří ; Pospíšek, Martin (advisor) ; Fišer, Radovan (referee)
Translation initiation is an important step in control of gene expression. Cellular mRNAs are usually translated via classical cap-dependent pathway, but some of them can also be translated by an alternative pathway like IRES mediated one. In the first part of my work I studied a possibility of IRES dependent translation initiation of RRN3 and BAS1 mRNAs from yeast Saccharomyces cerevisiae. I inserted BAS1 and RRN3 5'UTR into the intercistronic spacer of pFGAL4h and its promotreless version and measured the activity of secondary reporters in enegineered yeast strains. My data indicates presence of cryptic promoter in tested regions. To confirm this results I first inserted the RRN3 5'UTR into the intercistronic region of another bicistronic plasmid - pRFh and measured expression of second cistron. The results were similar like in the case of pFGAL4h system. The presence of shorter RNAs transcribed from cryptic promoter within the intercistronic region was shown using real time PCR. My quest in the second part of this work was to prepare RNAs useful for next investigation of translation control of lariat capped RNAs produced by GIR1 ribozyme in a slime mold Didymium iridis. I have prepared a wide set of DNA molecules coding different versions of GIR1 in front of the firefly luciferase ORF which served as...
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Transcriptional regulation of miR-17-92 microRNA cluster during macrophage differentiation.
Rybářová, Jana ; Stopka, Tomáš (advisor) ; Pospíšek, Martin (referee)
miR-17-92 cluster (Oncomir1) encodes seven microRNAs (miRNA, miR) regulating many biological processes including proliferation, differentiation or apoptosis. Overexpression of microRNAs encoded by miR-17-92 cluster is found in a number of tumors including acute and chronic myeloid leukemias (Dixon-McIver et al., 2008; Li et al., 2008; Venturini et al., 2007). Myeloid progenitors express miR-17-92 cluster at a high level, while macrophage differentiation associates with its downregulation. Our laboratory found, that miR-17-92 cluster is repressed by transcription factor Early growth response 2 (Egr2) upon differentiation of primary myeloid PUER progenitors, induced with transcription factor PU.1. Aim of this thesis is to further test the abovementioned data by preparing a reporter vectors set, carrying various fragments of miR-17-92 putative promoter, which enables us to study regulation of transcription of miR-17-92 cluster. This task complicated by presence of increased GC content of the miR-17-92 promoter was successfully accomplished resulting in amplification of eight fragments containing the various parts of miR-17-92 promoter including region -3.3 to 0 kb relative to the start of miR-17-5p sequence, that were inserted into pGL3 reporter vector. Transfection of pGL3 reporter vector carrying...
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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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