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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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Improvement of retroviral vectors for efficient gene transfer
Šenigl, Filip
44 CONCLUSIONS Retroviral vectors are transcriptionally unstable in mammalian cells. The ASLV- derived vectors are the most affected by silencing. We modified the vector by insertion of the CpG island inner element (IE) into the vector LTR in three different positions in either sense or antisense orientation. Each vector variant exhibited a certain extent of stabilization. The vector with insertion of a tandem of two IEs between the enhancer and the promoter was the most stable and exhibited almost no silencing after four months of cultivation in rodent and human cells. The IE comprises two high-affinity Sp1 binding sites. The presence of Sp1 binding sites is important for the protective effect of IE, but at least a part of the entire anti-silencing capacity is maintained in IE with mutated Sp1 sites. We identified the Tvc receptor of ASLV. The tvc gene encodes a 488-amino-acid protein most closely related to mammalian butyrophilins, members of the immunoglobulin protein family. To confirm the identification of the Tvc receptor, we disrupted both tvc alleles in normally susceptible DT40 cells. The DT40 tvc-/- clone was resistant to the ASLV(C) infection. We identified the mutation that results in decreased susceptibility to infection by ASLV subgroups B and D and resistance to ASLV subgroup E of line M...
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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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Improvement of retroviral vectors for efficient gene transfer
Šenigl, Filip
44 CONCLUSIONS Retroviral vectors are transcriptionally unstable in mammalian cells. The ASLV- derived vectors are the most affected by silencing. We modified the vector by insertion of the CpG island inner element (IE) into the vector LTR in three different positions in either sense or antisense orientation. Each vector variant exhibited a certain extent of stabilization. The vector with insertion of a tandem of two IEs between the enhancer and the promoter was the most stable and exhibited almost no silencing after four months of cultivation in rodent and human cells. The IE comprises two high-affinity Sp1 binding sites. The presence of Sp1 binding sites is important for the protective effect of IE, but at least a part of the entire anti-silencing capacity is maintained in IE with mutated Sp1 sites. We identified the Tvc receptor of ASLV. The tvc gene encodes a 488-amino-acid protein most closely related to mammalian butyrophilins, members of the immunoglobulin protein family. To confirm the identification of the Tvc receptor, we disrupted both tvc alleles in normally susceptible DT40 cells. The DT40 tvc-/- clone was resistant to the ASLV(C) infection. We identified the mutation that results in decreased susceptibility to infection by ASLV subgroups B and D and resistance to ASLV subgroup E of line M...
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Improvement of retroviral vectors for efficient gene transfer
Šenigl, Filip ; Hejnar, Jiří (advisor) ; Šmahel, Michal (referee) ; Ruml, Tomáš (referee)
44 CONCLUSIONS Retroviral vectors are transcriptionally unstable in mammalian cells. The ASLV- derived vectors are the most affected by silencing. We modified the vector by insertion of the CpG island inner element (IE) into the vector LTR in three different positions in either sense or antisense orientation. Each vector variant exhibited a certain extent of stabilization. The vector with insertion of a tandem of two IEs between the enhancer and the promoter was the most stable and exhibited almost no silencing after four months of cultivation in rodent and human cells. The IE comprises two high-affinity Sp1 binding sites. The presence of Sp1 binding sites is important for the protective effect of IE, but at least a part of the entire anti-silencing capacity is maintained in IE with mutated Sp1 sites. We identified the Tvc receptor of ASLV. The tvc gene encodes a 488-amino-acid protein most closely related to mammalian butyrophilins, members of the immunoglobulin protein family. To confirm the identification of the Tvc receptor, we disrupted both tvc alleles in normally susceptible DT40 cells. The DT40 tvc-/- clone was resistant to the ASLV(C) infection. We identified the mutation that results in decreased susceptibility to infection by ASLV subgroups B and D and resistance to ASLV subgroup E of line M...
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