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The role of Nuclear Phosphatidylinositol 4,5 bisphosphate in RNA Polymerase II Transcription
Balaban, Can ; Hozák, Pavel (advisor) ; Šebesta, Marek (referee) ; Castano, Enrique (referee)
(in English) The phosphatidylinositols are a subclass of glycerophospholipids with their inositol head group linked to the diacylglycerol backbone. The differential phosphorylation of the inositol head group yields seven different phosphoinositide phosphates (PIPs) which can be mono-, bis,- or tris-phosphorylated. The roles of the cytoplasmic PIPs have been extensively studied in vesicular trafficking, ion channels, generating second messengers and, membrane and cytoskeletal dynamics. While their cytoplasmic functions are very well described, the molecular mechanism of their nuclear functions are still poorly understood. From the nuclear PIPs, the Phosphatidylinositol 4,5-bisphosphate (PIP2) is the most abundant phosphoinositide in the cell nucleus and it participates to the nuclear architecture by regulating processes such as chromatin remodeling, DNA-damage response and gene expression. In the cell nucleus, it localizes mostly to nuclear speckles where it interacts with the splicing machinery. In nucleolus, PIP2 is involved in the RNA Polymerase I machinery to regulate rDNA transcription. Recently, we have defined a nucleoplasmic pool of PIP2 which is observed in 40 to 100nm foci. The nascent transcripts of RNA Polymerase II (RNAPII) were visualized at their periphery and RNA was shown to be...
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Factors important for Cajal body formation
Roithová, Adriana ; Staněk, David (advisor) ; Valentová, Anna (referee)
This research describes the structure and function of nuclear domains called Cajal bodies (CB). CB contain proteins and factors involved in assembly and modification of snRNPs. These bodies are found in vertebrates and invertebrates and even plants. Not all cell types contain CB. Their number and size depends on the transcription activity of cell and cell cycle phase. This paper discusses the factors that affect the CB formation. One of the most important factors is the level of snRNPs and transcription activity. Recently shows that an important role in CB formation has coilin and other components phosphorylation. Other works show the influence of the environment. There is also discussion regulation of CB biogenesis, witch is not yet fully understood. Key words: Cajal bodies, coilin, cell nucleus, snRNP, pre-mRNA splicing, transcription
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Localization matters: function of paxillin and phopholipids in the cell nucleus
Marášek, Pavel ; Hozák, Pavel (advisor) ; Půta, František (referee) ; Žárský, Viktor (referee)
(English) Both paxillin and PIP2 are well known components of the cell, although of a distinct origin. Focal adhesion protein paxillin spreads the signals from extracellular matrix via integrins and growth factor receptors to affect cellular motility and migration (Schaller, 2001). PIP2, a major structural component of cytoplasmic membrane, is utilized by phospholipase C to generate second messenger molecules (Hokin and Hokin 1953; Streb et al. 1983). Both molecules were recently shown to be localized in the nucleus. Their original functions have been well established, but together with other research colleagues we are now shedding more light on completely different functions of these biological molecules and moreover, in the different compartments than they were primarily believed to function in. Here, we introduce paxillin as an important factor of the cell nucleus, where it regulates transcription of two important growth-related genes, IGF2 and H19. It does not affect the allelic expression of these imprinted genes, it rather regulates long-range chromosomal interactions between H19 or IGF2 promoter, and the shared distal enhacer on an active allele. In detail, paxillin stimulates the interaction between the enhancer and the IGF2 promoter, activating IGF2 gene transcription, while it restrains...
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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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Analysis of functional interactions of phospholipids in the cell nucleus.
Biddle, Veronika ; Hozák, Pavel (advisor) ; Kaňka, Jiří (referee) ; Malínský, Jan (referee)
(English) Phosphoinositides (PIs) are glycerophospholipids with a negative charge. As components of cell membranes, PIs are involved in membrane and cytoskeletal dynamics, cell movement and signalling, and the modulation of ion channels and transporters. Apart from the cytoplasm, phosphoinositides also localise to the cell nucleus. PIs play a role in crucial nuclear processes, such as DNA transcription, pre-rRNA and pre-mRNA processing, cell differentiation, DNA damage response, or apoptosis. Phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are the most abundant phosphoinositides in the cell. However, their exact localisation and function in the nucleus are largely unknown. Here, we describe their localisation at super-resolution level and their involvement in some nuclear processes. PI(4)P is present in nuclear lamina, nuclear speckles and nucleoli, and it forms small foci in nucleoplasm. The majority of nuclear PI(4)P localises to the nucleoplasm, whereas almost 16 % is present in nuclear speckles. On the other hand, the majority of nuclear PI(4,5)P2 localises to nuclear speckles, almost 30 % localises to nucleoplasm and the lesser portion to nucleoli. In the nucleoplasm, PI(4,5)P2 forms small foci called nuclear lipid islets (NLIs). Their core is...
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Virus translocation from cytoplasm to the cell nucleus
Išler, Lukáš ; Forstová, Jitka (advisor) ; Přikryl, David (referee)
Replication cycles of most DNA and some RNA viruses require translocation of these viruses into the host cell nucleus. In this step viruses must cross the nuclear membrane. In the cell cycle interphase, it can be exceeded only by passing through nuclear pores. Individual virus families have developed different strategies to efficiently translocate through the nuclear pore. In this paper, summarizing the knowledge of viral penetration through the nuclear pore, it is shown that in addition to the interaction with transport receptors, viral particles interact directly with some proteins of the nuclear pore complex, called nucleoporins (NUPs). Especially, one group of nucleoporins, so-called FG NUPs, interacts with viral particles. Their sequence contains naturally disordered domains rich in phenylalanine-glycine (FG) repeats which create selective barrier of the nuclear pore complex. These are mainly nucleoporins NUP153, NUP214 and NUP358. Interaction of viral particles with these nucleoporins allow them to cross this barrier and deliver their viral genome to the host cell nucleus. It is therefore an essential step in the early phase of the viral infection cycle. Keywords: cell nucleus, nuclear transport, nuclear pore complex, nucleoporins, virus nuclear entry, NUP153, NUP214, NUP358
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Function of nuclear phosphoinositides and their binding partners in gene expression
Uličná, Lívia ; Hozák, Pavel (advisor) ; Šolc, Petr (referee) ; Macůrková, Marie (referee)
(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids with inositol head (ring) which can be phosphorylated. Inositol ring phosphorylation yields in seven different PIs species which can be mono-, bis,- or tris-phosphorylated. Roles of cytoplasmic PIs have been extensively studied in for membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generating of second messengers. Nuclear PIs have been implicated in posttranscriptional processing of pre-mRNA, DNA transcription and chromatin remodelling. While cytoplasmic functions are very well described, the molecular mechanism of their nuclear functions are still poorly understood. In this study we focus on description of localization of nuclear PIs in particular functional nuclear compartments, which enable us to reveal PIs involvement in nuclear processes. We also focused on identification of nuclear PIs involved in the regulation of genes transcription and revealed detailed mechanism of PI(4,5)P2 a PHF8 interaction in the regulation of ribosomal genes transcription. By two independent approaches, we have described PIs localization to the nuclear membrane, nuclear speckles, small foci in the nucleoplasm, and the nucleolus. This spread nuclear localization suggests and confirms PI's...
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Gene regulation in four dimensions
Vaňková Hausnerová, Viola ; Lanctôt, Christian (advisor) ; Převorovský, Martin (referee) ; Krásný, Libor (referee)
Transcription has turned out to be a discontinuous process when imaged at a single cell level. This observation is referred to as transcriptional bursting or pulsing and has been detected in a variety of organisms ranging from bacteria to mammalian cells. The dynamics of transcriptional pulsing are influenced by the properties intrinsic to the transcriptional process, as well as by upstream factors: chromatin environment, signalling molecules, cell cycle stage etc. In the first part of this thesis, we focused on the regulation of transcriptional pulsing in the nucleolus. Using imaging of living cells, we detected pulsatile transcription of a transgene with nucleolar localization whose expression was mediated by RNA polymerase II. In the second part of the thesis, we investigated the relationship between chromatin decondensation and transcriptional dynamics. We used hyperosmotic medium to induce global condensation of chromatin and revealed that upon chromatin decondensation, a transient spike in transcriptional intensity occurs in induvial living cells. Next, we analysed expression of TFRC and POLR2A genes in several cell cycle stages using single molecule RNA FISH. We detected increase in both frequency and size of transcriptional pulses during a limited time window which coincided with chromatin...
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Localization and function of phosphoinositides in the cell nucleus
Kalasová, Ilona ; Hozák, Pavel (advisor) ; Stopka, Tomáš (referee) ; Šolc, Petr (referee)
(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids. Their inositol head can be phosphorylated at three positions generating seven differently phosphorylated species. Cytoplasmic phosphoinositides regulate membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generate second messengers. In the nucleus, PIs are implicated in pre-mRNA processing, DNA transcription and chromatin remodelling. However, their nuclear functions are still poorly understood. Here we focus on nuclear phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). We describe their localization and interaction with proteins involved in regulation of DNA transcription. PI(4)P localizes to nuclear membrane, nuclear speckles and nucleoplasm. The majority of nuclear PI(4)P is associated with chromatin and colocalizes with H3K4me2. PI(4,5)P2 localizes to nucleoli and nuclear speckles. Besides, 30 % of nuclear PI(4,5)P2 forms small nucleoplasmic PI(4,5)P2 islets. They have carbon rich core, which is probably formed by lipids, and are surrounded by proteins and nucleic acids. The active form of RNA polymerase II associates with PI(4,5)P2 islets and DNA is actively transcribed in the vicinity of PI(4,5)P2 islets. Moreover,...
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Formation of splicing machinery in the context of the cell nucleus
Stejskalová, Eva ; Staněk, David (advisor) ; Vanáčová, Štěpánka (referee) ; Malínský, Jan (referee)
Most of the protein coding genes of higher eukaryotes contain introns which have to be removed from primary transcripts to make mRNA which can be used as a template for protein synthesis. This crucial step in the pre-mRNA processing is carried out by the spliceosome, a complex ribonucleoprotein machine formed from small ribonucleoprotein particles (snRNPs). snRNPs biogenesis is a complex process composed of several steps which take place in both the cytoplasm and the nucleus. Spliceosome assembly is highly dynamic and tightly regulated and pre-mRNA splicing depends not only on the sequence of the pre-mRNA itself but also on the nuclear context, such as the chromatin modifications. How do cells regulate where and when the spliceosome would be assembled? What determines which introns will be spliced? These are fundamental, yet unanswered, biological questions. In this work we analyzed the formation of splicing machinery in the context of the cell nucleus from several different points of view. First, we investigated the unexpected connection between splicing factor U1-70K and the survival of motor neurons (SMN) complex which is a major player in the snRNP biogenesis pathway. We revealed that U1-70K interacts with the SMN complex and that this interaction is crucial for the stability of nuclear gems, small...
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