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The relationship between chromatin organization and RNA splicing
Icha, Jaroslav ; Staněk, David (advisor) ; Valentová, Anna (referee)
It is well known that RNA splicing and other pre-mRNA processing reactions happen cotranscriptionally. Surprisingly, there were recently discovered some chromatin features that had uneven distribution between exons and introns, which directly links chromatin organisation to splicing. This work summarizes all the studies that detected these chromatin patterns on exons and discuss their inconsistencies. In these studies nucleosomes were found to be preferentially positioned on exons, specific histone modifications and DNA methylation were also enriched on exons. These local patterns of chromatin organisation were evolutionarily conserved from mammals (human and mouse) to worm C. elegans and fly D. melanogaster. Their findings indicate that the role for chromatin structure in pre-mRNA splicing is to promote exon recognition. There are two mechanisms proposed for this role of chromatin in splicing. The first one is influence on RNA polymerase II elongation speed, and the second is specific recruitment of splicing machinery. In the near future we can expect studies searching for concrete examples of these two mechanisms and assessing their significance. Indeed it was reported very recently that H3K36me3 regulates alternative splicing via the second mechanism.
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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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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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|
|
The relationship between chromatin organization and RNA splicing
Icha, Jaroslav ; Valentová, Anna (referee) ; Staněk, David (advisor)
It is well known that RNA splicing and other pre-mRNA processing reactions happen cotranscriptionally. Surprisingly, there were recently discovered some chromatin features that had uneven distribution between exons and introns, which directly links chromatin organisation to splicing. This work summarizes all the studies that detected these chromatin patterns on exons and discuss their inconsistencies. In these studies nucleosomes were found to be preferentially positioned on exons, specific histone modifications and DNA methylation were also enriched on exons. These local patterns of chromatin organisation were evolutionarily conserved from mammals (human and mouse) to worm C. elegans and fly D. melanogaster. Their findings indicate that the role for chromatin structure in pre-mRNA splicing is to promote exon recognition. There are two mechanisms proposed for this role of chromatin in splicing. The first one is influence on RNA polymerase II elongation speed, and the second is specific recruitment of splicing machinery. In the near future we can expect studies searching for concrete examples of these two mechanisms and assessing their significance. Indeed it was reported very recently that H3K36me3 regulates alternative splicing via the second mechanism.
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