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The role of acetylation in the RNA recognition motif of SRSF5 protein
Icha, Jaroslav ; Staněk, David (advisor) ; Šenigl, Filip (referee)
Acetylation is emerging as an important posttranslational modification, which is found in thousands of proteins in eukaryotes, as well as prokaryotes. Global proteomic studies implicated acetylation in regulation of various processes like metabolism, gene expression, cell cycle or aging to name a few. In this work I set out to investigate the role of acetylation of a splicing regulatory protein SRSF5 by creating mutations in its acetylation site. I tested the hypothesis that acetylation influences SRSF5 interaction with RNA. I expressed acetylation-mimicking (Q) or non-acetylable (R) mutant of SRSF5 in HeLa cells and measured their interaction with RNA by RNA immunoprecipitation or in vitro by fluorescence anisotropy. Both approaches agreed that mutants interact with RNA less than the wild type protein and Q mutant bound RNA weaker than R mutant. I did not detect further difference in localization or dynamics among the proteins in vivo, which suggests that difference caused by weakened interaction of mutants with RNA was outweighed by other factors influencing SRSF5 behaviour, probably protein-protein interactions. I also found out that mutant SRSF5 proteins do not have a dominant effect on splicing of fibronectin alternative EDB exon. The data obtained give an indirect evidence for the hypothesis that...
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Regulation of alternative splicing
Dušková, Eva ; Staněk, David (advisor) ; Trejbalová, Kateřina (referee)
Alternative splicing is an important cellular mechanism. It allows to produce multiple protein isoforms from a limited number of genes. Regulation of alternative splicing involves cis-acting elements on pre-mRNA and trans-acting splicing factors (SR and hnRNP proteins). Because splicing occurs co-transcriptionaly, chromatin structure appears to have a role in the regulation of alternative splicing. We have studied the effect of histone acetylation on alternative splicing. We have prepared splicing reporter for alternative EDB exon, which is part of the fibronectin gene. We have shown, that the inhibition of histone deacetylases affects splicing pattern of EDB exon from the reporter in the same way as the splicing of the endogenous EDB exon. Furthermore, we have shown, that the structure of the promoter affects splicing of alternative EDB exon from splicing reporter. Currently we have found out, that the structure of the promoter influences the degree of histone H4 acetylation. Inclusion of alternative EDB exon in mRNA was inversely proportional to histon acetylation on the reporter. This work might explain why various promoters have different splicing patterns of alternative exons.
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Analýza regulace komplexů cytoplazmatických poly(A) polymeráz
Novák, Jakub ; Staněk, David (advisor) ; Hrossová, Dominika (referee)
The regulation of gene expression is achieved at many levels. Chromatin-based gene regulation has been the central focus of many decades of research; however, posttranscriptional control mechanisms are emerging as a fundamental complement to direct protein synthesis. This thesis is focused on a specific mechanism of posttranscriptional control - the translational regulation of mRNAs in the cell cytoplasm. This control is a consequence of the balance between translational repression and activation and hinges on the selective recognition of regulated mRNAs by RNA-binding proteins and their ability to recruit RNA modifying proteins. In this thesis, Caenorhabditis elegans germline was used to study translational control of the germ cell-enriched gene, gld-2. Mutants of known RNA-binding proteins of the PUF and CPB protein families were analyzed by performing Western blots, using anti-GLD-2 antibodies. Yeast 3-Hybrid system was used to identify the cis-regulatory sites in the gld-2 mRNA conferring translational regulation by members of PUF and CPB protein families. Potential autoregulatory loop of gld-2 gene expression was also investigated. This thesis shows that FBF proteins positively regulate expression of gld-2 and bind to a conserved sequence in the 3'UTR of its mRNA. Mutations of gld-2 negatively affect...
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Determinants of the splice site selection in protein-coding and long non-coding RNAs
Krchňáková, Zuzana ; Staněk, David (advisor) ; Svoboda, Petr (referee) ; Blažek, Dalibor (referee)
In my thesis, I focused on several underexplored areas of RNA splicing regulation. In the first part, I analyzed how chromatin and transcription regulatory elements change pre-mRNA splicing. In the second part, I studied why long non-coding RNAs (lncRNAs) are spliced less efficiently than protein-coding mRNAs. Finally, I was testing the importance of intron for the activating function of lncRNAs. It has been shown that chromatin and promoter identity modulate alternative splicing decisions. Here, I tested whether local chromatin and distant genomic elements that influence transcription can also modulate splicing. Using the chromatin modifying enzymes directly targeted to FOSL1 gene by TALE technology, I showed that changes in histone H3K9 methylation affect constitutive splicing. Furthermore, I provide evidence that deletion of transcription enhancer located several kilobases upstream of an alternative exons changes splicing pattern of the alternative exon. Many nascent lncRNAs undergo the same maturation steps as pre-mRNAs of protein- coding genes (PCGs), but they are often poorly spliced. To identify the underlying mechanisms for this phenomenon, we searched for putative splicing inhibitory sequences. Genome-wide analysis of intergenic lncRNAs (lincRNAs) revealed that, in general, they do not...
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Structure and characteristics of mutant hPrp31 in Retinitis pigmentosa
Těšina, Petr ; Staněk, David (advisor) ; Lišková, Petra (referee)
Retinitis pigmentosa is a hereditary eye disease causing progressive loss of photoreceptor cells, which leads to an irreversible sight handicap and eventually complete blindness. It is a major cause of visual handicap or blindness with very heterogenous genetic background. There are four genes accounting for retinitis pigmentosa that encode splicing factors necessary for spliceosomal assembly and function. Unlike any of the other known genes associated with this disease, these are all expressed ubiquitously throughout the human body. Intriguingly, the mutant forms of these vital splicing factors cause cell-type specific disease affecting only photoreceptor cones and rods. Molecular mechanisms underlying this cell-type specific effect remain elusive. One of these splicing factors is the hPrp31 protein. Its mutant form known as AD29 is the focal point of this thesis. Some of the effects of this mutation on the cellular level have been discovered recently. A creation of the expression vector followed by expression and purification of the truncated hPrp31 protein carrying the AD29 mutation is presented in this thesis. The purified product has been used for production of a αhPrp31 polyclonal rabbit antibody, whose applicability to western blot and immunofluorescence staining has been verified. Moreover...
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The role of coilin in snRNP quality control
Kuzmenko, Darya ; Staněk, David (advisor) ; Abrhámová, Kateřina (referee)
Mammalian genes are transcribed as precursors - pre-mRNA. They contain coding sequences (exons) and non-coding sequences (introns). Splicing, a process of cutting out introns and joining exons to generate mature mRNA, is carried out by a spliceosome. The spliceosome consists of five small nuclear ribonucleoprotein (snRNP) particles and numerous associated proteins. Its assembly is a complex process involving a specific nuclear sub-compartment, the Cajal body (CB). Here, we investigate function of the CB scaffold protein, coilin, in snRNP quality control in HeLa cells. Sequestration of immature snRNP in coilin-deficient cells is analysed by fluorescence in situ hybridisation. We show that without coilin the cells are unable to sequester them. Next, we provide evidence that absence of coilin does not sensitise HeLa cells for perturbation in snRNP maturation in terms of cell proliferation. Moreover, coilin deficiency does not result in significant changes in U4, U5 or U6 snRNA steady state levels. Therefore, coilin, and, in this way, Cajal bodies do not become essential under the conditions of strained snRNP biogenesis.
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Molecular mechanism of quality control during snRNP biogenesis
Klimešová, Klára ; Staněk, David (advisor) ; Krásný, Libor (referee) ; Vomastek, Tomáš (referee)
The spliceosome is one of the largest and most dynamic molecular machines in the cell. The central part of the complex is formed by five small nuclear ribonucleoproteins (snRNPs) which are generated in a multi-step biogenesis pathway. Moreover, the snRNPs undergo extensive rearrangements during the splicing and require reassembly after every intron removal. Both de novo assembly and post-splicing recycling of snRNPs are guided and facilitated by specific chaperones. Here, I reveal molecular details of function of two snRNP chaperones, SART3 and TSSC4. While TSSC4 is a previously uncharacterized protein, SART3 has been described before as a U6 snRNP-specific factor which assists in association of U6 and U4 particles into di-snRNP, and is important for the U4/U6 snRNP recycling. However, the mechanism of its function has been unclear. Here, I provide an evidence that SART3 interacts with a post-splicing complex and propose that SART3 could promote its disassembly. Our data further suggest that SART3 binds U6 snRNP already within the post-splicing complex and thus participates in the whole recycling phase of U6 snRNP. Then, I show that TSSC4 is a novel U5 snRNP-specific chaperone which promotes an assembly of U5 and U4/U6 snRNPs into a splicing-competent tri-snRNP particle. We identified...
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