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Replication and transcription of nucleolar DNA
Flusser, Michal ; Smirnov, Evgeny (advisor) ; Staněk, David (referee)
The nucleolus is the most prominent compartment of the cell nucleus and is the place where ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins. Although the nucleolus has been studied for decades its structural and functional organization is still unclear. In particular, the role of various types of DNA participating in the formation of nucleoli along with ribosomal genes is not understood. The present thesis summarizes the current knowledge and views on the nucleolus, focusing on the two synthetic processes, replication and transcription, in mammalian cells. Specific features of these processes in the context of nucleolar ultrastructure remains an unsolved problem of the modern molecular biology.
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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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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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Recycling of spliceosomal complexes
Klimešová, Klára ; Staněk, David (advisor) ; Hálová, Martina (referee)
Most human genes are composed of coding sequences (exons) that are interrupted by non-coding sequences (introns). After gene transcription into pre-mRNA, these introns have to be removed in a process called splicing. Splicing is mediated by a very complex and dynamic complex called the spliceosome, which consists of five small nuclear ribonucleoprotein particles (snRNPs) and numerous additional splicing proteins. Each particle contains single small nuclear RNA and a set of specific proteins. SnRNPs are assembled by a stepwise process that takes place both in the nucleus and the cytoplasm and final maturation steps occur in nuclear Cajal bodies. The mature snRNPs interact with pre-mRNA in an ordered pathway and form the spliceosome that catalyzes two trans-esterification reactions leading to intron excision and exons ligation. Subsequently, the spliceosome disassembles again into individual snRNPs that have undergone diverse conformational and compositional transformations during splicing. Thus, before the particles can participate in another round of splicing they have to go through recycling to recover their original form. However, currently the recycling phase of the splicing cycle is surrounded by more questions than answers. The purpose of this work is to discuss latest findings that shed some light on...
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Chromatin modifiers and their relation to transcription regulation in Saccharomyces cerevisiae
Hálová, Martina ; Folk, Petr (advisor) ; Staněk, David (referee)
Relations among transcription, pre-mRNA processing and chromatin modifications are only partially understood. The human protein SNW1/SKIP belongs to factors which couple these processes. The protein plays role in pre-mRNA splicing and transcription on the level of both initiation and elongation. According to the hypothesis of K. Jones laboratory, it physically and functionally interacts with positive transcription elongation factor b during transcription elongation and influences methylation of histone H3 on lysine 4, a modification characteristic for active transcription (Bres et al., Genes Dev. 19:1211-26, 2005, Bres et al., Mol Cell. 36:75-87, 2009). The yeast ortholog of SNW1/SKIP, Prp45, was until now reported only in connection with splicing regulation. However, unpublished results from our Laboratory and others showed that it is employed in transcription elongation as well. The aim of the diploma project was to search for the relations between Prp45 and the factors regulating transcription. It was confirmed that the mutation prp45(1 169) results in the delay of PHO5 and PHO84 expression during transcriptional induction. Next, we discovered new genetic interactions between PRP45 and several genes encoding the effectors of chromatin modifications. How Prp45 influences the expression of PHO5 and PHO84...
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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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