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A Multistructure Created By Coverings Of A Set
Staňek, David
This paper focuses on a certain construction of a multistructure on the set of all coverings on the universal set U and discusses properties of this multistructure. The construction itself uses a concept dual to the Ends Lemma, called Beginnings Lemma, which is proved in the paper.
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Quality control in snRNP biogenesis
Roithová, Adriana ; Staněk, David (advisor) ; Malínský, Jan (referee) ; Vomastek, Tomáš (referee)
(English) snRNPs are key components of the spliceosome. During their life, they are found in the cytoplasm and also in the nucleus, where carry out their function. There are five major snRNPs named according to RNA they contain U1, U2, U4, U5 and U6. Each snRNP consists from RNA, ring of seven Sm or LSm proteins and additional proteins specific for each snRNP. Their biogenesis starts in the nucleus, where they are transcribed. Then they are transported into the cytoplasm. During their cytoplasmic phase, the SMN complex forms the Sm ring around the specific sequence on snRNA and cap is trimethylated. These two modifications are the signals for reimport of snRNA into the nucleus, where they accumulate in the nuclear structures called Cajal bodies (CBs), where the final maturation steps occur. There are several quality control points during snRNP biogenesis that ensure that only fully assembled particles reach the spliceosome. The first checkpoint is in the nucleus immediately after the transcription, when the export complex is formed. The second checkpoint is in the cytoplasm and proofreads Sm ring assembly. If the Sm ring formation fails, the defective snRNPs are degraded in the cytoplasm by Xrn1 exonuclease. However, it is still unclear, how the cell distinguishes between normal and defective...
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Rough Sets on State Spaces of Automata
Staněk, David
This paper discussed a subclass of finite automata, which have ordering on the state sets created by a transition (or next-state) function. Hence, there do not exist cycles of more than one element. We discuss a relation of equality of upper closure on the systems of all subsets of state systems of quasi-automata, which creates an equivalence.
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Influence of transcription regulatory elemets on pre-mRNA splicing
Volek, Martin ; Staněk, David (advisor) ; Malík, Radek (referee)
In the process of pre-mRNA splicing introns are removed from pre-mRNA and exons are joined together. Current studies show, that about 95 % of genes, which contain more than two exons, can undergo alternative splicing. In this process some exons are included in or excluded from the final mRNA. Majority of pre-mRNA splicing take place co- transcriptionaly at this time RNA polymerase II is still attached to pre-mRNA. Alternative splicing is complex process that takes place in a close proximity of DNA and histones that might modulate alternative splicing decisions. Futher studies have validated fibronectin gene (FN1) and his alternative exons EDA and EDB (extra domain A and B) as suitably model for studying alternative splicing. Study using FN1 minigene reporter system, which is composed from EDA exon and two surrounding introns and exons, has proved that insertion of transcription enhancer SV40 infront of promotor, the level of EDA inclusion is decreased. So far, has not been prooved if this mechanism can function in real genome context and if distal transcription elements can influence alternative splicing. In this study, we have predicted transcription enhancer for FN1 gene by using The Ensemble Regulatory Build and FANTOM 5. The predicted transcription enhancer, is located 23,5 kbp upstream of TSS...
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Importance of 5'-end structures of eukaryotic mRNA molecules
Vopálenský, Václav ; Pospíšek, Martin (advisor) ; Lukeš, Julius (referee) ; Staněk, David (referee)
73 V. SHRNUTÍ VÝSLEDKŮ IRESITE: THE DATABASE OF EXPERIMENTALLY VERIFIED IRES STRUCTURES (WWW.IRESITE.ORG) IRESite je kurátorovaná relační databáze typu MySQL-4.1 využívající InnoDB tabulky. Do databáze je možné vložit položky dvojího typu: o natural položky obsahují veškerá experimentální data týkající se určité IRES struktury (kompletní sekvence mRNA s vymezením pozic IRES sekvence a kódovaného proteinu, veškeré proteiny interagující s IRES, případná sekundární struktura) o engineered položky popisují uměle vytvořené plazmidy. Většinou se jedná o bicistronní plazmidy sloužící k testování funkčnosti sekvence, o níž se předpokládá, že obsahuje IRES element, a případné mutantní deriváty této sekvence. Do IRESite se v tomto případě vkládají údaje o sekvenci mRNA s vymezením IRES oblasti, informace o reportérových proteinech, informace o translačních experimentech včetně použitého promotoru a informace o pozitivních a negativních kontrolách. V databázi je nyní uloženo 288 položek; 67 natural (20 virových a 47 buněčných) a 221 engineered. IRESite slouží nejen k ukládání experimentálních dat, ale umožňuje také jejich prohledávání podle klíčových slov, případně následné porovnávání vybraných experimentů. A BIOINFORMATICAL APPROACH TO THE ANALYSIS OF VIRAL AND CELLULAR INTERNAL RIBOSOME ENTRY Publikace...
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Molecular principles of translation reinitiation in mammals
Hronová, Vladislava ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Staněk, David (referee)
Translation initiation is a multistep process resulting in the formation of the elongation-competent 80S ribosome at the AUG start codon of the mRNA to be translated into a polypeptide chain. This process is orchestrated by numerous proteins called eukaryotic initiation factors (eIFs), out of which the most multitasking one is the eukaryotic initiation factor 3 (eIF3). The main focus of our laboratory aims at the complex characterization of the multisubunit protein eIF3 and the mechanisms of its contribution to various steps of translation initiation. Besides this, we also study one of the gene-specific translational control mechanisms called reinitiation which was, at least in yeast, also shown to be promoted by eIF3. Here I show that the N-terminal domain (NTD) of the largest subunit of yeast eIF3, a/Tif32, plays an important role not only in anchoring the eIF3 complex to the 40S small ribosomal subunit but it also critically contributes to mRNA recruitment to the 43S preinitiation complexes in vivo. The mRNA stabilization role of the a/Tif32-NTD at the mRNA exit channel of the 40S subunit was further confirmed in our following study by biophysical experiments. There, using in vivo approaches, we also demonstrated that mRNAs with longer 5'UTRs are more dependent on the stabilization role of the...
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The role of pre-mRNA splicing in human hereditary diseases
Malinová, Anna ; Staněk, David (advisor) ; Vanáčová, Štěpánka (referee) ; Krásný, Libor (referee)
U5 small ribonucleoprotein particle (U5 snRNP) is a crucial component of the spliceosome, the complex responsible for pre-mRNA splicing. Despite the importance of U5 snRNP, not much is known about its biogenesis. When we depleted one of the core U5 components, protein PRPF8, the other U5-specific proteins do not associate with U5 snRNA and the incomplete U5 was accumulated in nuclear structures known as Cajal bodies. To further clarify the role of PRPF8 in U5 snRNP assembly, we studied PRPF8 mutations that cause an autosomal dominant retinal disorder, retinitis pigmentosa (RP). We prepared eight different PRPF8 variants carrying RP-associated mutations and expressed them stably in human cell culture. We showed that most mutations interfere with the assembly of snRNPs which consequently leads to reduced efficiency of splicing. The mutant PRPF8 together with EFTUD2 are stalled in the cytoplasm in a form of U5 snRNP assembly intermediate. Strikingly, we identified several chaperons including the HSP90/R2TP complex and ZNHIT2 as new PRPF8's interactors and potential U5 snRNP assembly factors. Our results further imply that these chaperons preferentially bind the unassembled U5 complexes and that HSP90 is required for stability of...
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The role of translation initiation factor 3 (eIF3) in translation termination.
Beznosková, Petra ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Staněk, David (referee)
Protein synthesis is a tightly regulated process of gene expression. Each gene has its start and its stop, which is determined by one of the three stop codons. Many recent articles describe ribosomes that purposely bypass stops on specific mRNAs to extend the nascent polypeptide to alter its properties. It is called programmed stop codon readthrough. Since over 15% of human genetic diseases are caused by so called premature termination codons (PTC) that halt translation and produce truncated proteins, this mechanism has a great potential implication in medical research. Numerous labs search for non-toxic drugs specifically increasing readthrough at PTCs; however, the success of this effort requires identification and understanding of all factors that are involved in this process. Here, we present one such factor eukaryotic initiation factor 3 (eIF3) and describe its ability to induce readthrough on stop codons in termination non-favorable context during programmed readthrough and also the consequences of its action on translation regulation. We additionally analyzed which near-cognate (nc) tRNAs are incorporated at UGA stop codons depending on the nucleotide that immediately follows them (so called +4 base). This way we established new rules for stop codon decoding and identified so called...
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Role of promoter in the regulation of alternative splicing
Kozáková, Eva ; Staněk, David (advisor) ; Půta, František (referee) ; Blažek, Dalibor (referee)
It was shown that 95 % of human multi-exon genes are alternatively spliced and the regulation of alternative splicing is extremely complex. Most pre-mRNA splicing events occur co- transcriptionally and there is increasing body of evidence, that chromatin modifications play an important role in the regulation of alternative splicing. Here we showed that inhibition of histone deacetylases (HDACs) modulates alternative splicing of ~700 genes via induction of histone H4 acetylation and increase of Pol II elongation rate along alternative region. We identified HDAC1 the catalytic activity of which is responsible for changes in alternative splicing. Then, we analyzed whether acetylhistone binding protein Brd2 regulates alternative splicing and showed that Brd2 occupies promoter regions of targeted genes and controls alternative splicing of ~300 genes. Later we showed that knockdown of histone acetyltransferase p300 promotes inclusion of the alternative fibronectin (FN1) EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as the p300 knockdown. Next we showed that p300 controls histone...
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