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Functional analysis of hPrp8 mutations linked to retinitis pigmentosa.
Matějů, Daniel ; Cvačková, Zuzana (advisor) ; Král, Vlastimil (referee)
hPrp8 is an essential pre-mRNA splicing factor. This highly conserved protein is a component of the U5 small ribonucleoprotein particle (U5 snRNP), which constitutes one of the building blocks of the spliceosome. hPrp8 acts as a key regulator of spliceosome activation and interacts directly with U5 snRNA and with the regions of pre-mRNA that are involved in the transesterification reactions during splicing. Mutations in hPrp8 have been shown to cause an autosomal dominant form of retinitis pigmentosa (RP), an inherited disease leading to progressive degeneration of retina. In this study, we analyzed the effects of the RP-associated mutations on the function of hPrp8. Using BAC recombineering, we created mutant variants of hPrp8-GFP construct and we generated stable cell lines expressing the recombinant proteins. The mutant proteins were expressed and localized to the nucleus. However, one of the missense mutations affected the localization and stability of hPrp8. Further experiments suggested that RP-associated mutations affect the ability of hPrp8 to interact with other components of the U5 snRNP and with pre-mRNA. We further studied the biogenesis of U5 snRNP. We depleted hPrp8 by siRNA to interfere with U5 snRNP assembly and we observed that the incompletely assembled U5 snRNPs accumulate in...
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Quality control in snRNP biogenesis
Roithová, Adriana ; Staněk, David (advisor) ; Malínský, Jan (referee) ; Vomastek, Tomáš (referee)
(English) snRNPs are key components of the spliceosome. During their life, they are found in the cytoplasm and also in the nucleus, where carry out their function. There are five major snRNPs named according to RNA they contain U1, U2, U4, U5 and U6. Each snRNP consists from RNA, ring of seven Sm or LSm proteins and additional proteins specific for each snRNP. Their biogenesis starts in the nucleus, where they are transcribed. Then they are transported into the cytoplasm. During their cytoplasmic phase, the SMN complex forms the Sm ring around the specific sequence on snRNA and cap is trimethylated. These two modifications are the signals for reimport of snRNA into the nucleus, where they accumulate in the nuclear structures called Cajal bodies (CBs), where the final maturation steps occur. There are several quality control points during snRNP biogenesis that ensure that only fully assembled particles reach the spliceosome. The first checkpoint is in the nucleus immediately after the transcription, when the export complex is formed. The second checkpoint is in the cytoplasm and proofreads Sm ring assembly. If the Sm ring formation fails, the defective snRNPs are degraded in the cytoplasm by Xrn1 exonuclease. However, it is still unclear, how the cell distinguishes between normal and defective...
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Study of the organization and dynamics of the membraneless cell compartments
Blažíková, Michaela
of Doctoral Thesis Title: Study of the organization and dynamics of the membraneless cell compartments Author: Michaela Blažíková Institute: Charles University in Prague, Faculty of Mathematics and Physics, Institute of Physics of Charles University Supervisor: Doc. RNDr. Petr Heřman, CSc., Charles University in Prague, Faculty of Mathematics and Physics, Institute of Physics of Charles University Abstract Eukaryotic cells contain many organelles and specific bodies. Beside the membrane delimited organelles such as nucleus, mitochondria or Golgi apparatus there are other structurally and functionally distinct membraneless structures in the cells. In this work we studied the self-organization processes, i.e. the processes that do not require specific interactions, of membraneless structures in nuclei, cytoplasm and plasma membrane of mammalian cells and yeast. The research was focused on the formation of nucleoli and Cajal bodies in mammalian cell nulei and processing bodies (P- bodies) in the cytoplasm of mammalian cells. The organization of MCC domains in the yeast plasma membrane (Membrane compartment of Can1) was studied as well. It was shown that nonspecific interactions as the result of macromolecular crowding could be one of the main driving forces in formation and stabilization of these...
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Quality control in snRNP biogenesis
Roithová, Adriana ; Staněk, David (advisor) ; Malínský, Jan (referee) ; Vomastek, Tomáš (referee)
(English) snRNPs are key components of the spliceosome. During their life, they are found in the cytoplasm and also in the nucleus, where carry out their function. There are five major snRNPs named according to RNA they contain U1, U2, U4, U5 and U6. Each snRNP consists from RNA, ring of seven Sm or LSm proteins and additional proteins specific for each snRNP. Their biogenesis starts in the nucleus, where they are transcribed. Then they are transported into the cytoplasm. During their cytoplasmic phase, the SMN complex forms the Sm ring around the specific sequence on snRNA and cap is trimethylated. These two modifications are the signals for reimport of snRNA into the nucleus, where they accumulate in the nuclear structures called Cajal bodies (CBs), where the final maturation steps occur. There are several quality control points during snRNP biogenesis that ensure that only fully assembled particles reach the spliceosome. The first checkpoint is in the nucleus immediately after the transcription, when the export complex is formed. The second checkpoint is in the cytoplasm and proofreads Sm ring assembly. If the Sm ring formation fails, the defective snRNPs are degraded in the cytoplasm by Xrn1 exonuclease. However, it is still unclear, how the cell distinguishes between normal and defective...
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Study of the organization and dynamics of the membraneless cell compartments
Blažíková, Michaela
of Doctoral Thesis Title: Study of the organization and dynamics of the membraneless cell compartments Author: Michaela Blažíková Institute: Charles University in Prague, Faculty of Mathematics and Physics, Institute of Physics of Charles University Supervisor: Doc. RNDr. Petr Heřman, CSc., Charles University in Prague, Faculty of Mathematics and Physics, Institute of Physics of Charles University Abstract Eukaryotic cells contain many organelles and specific bodies. Beside the membrane delimited organelles such as nucleus, mitochondria or Golgi apparatus there are other structurally and functionally distinct membraneless structures in the cells. In this work we studied the self-organization processes, i.e. the processes that do not require specific interactions, of membraneless structures in nuclei, cytoplasm and plasma membrane of mammalian cells and yeast. The research was focused on the formation of nucleoli and Cajal bodies in mammalian cell nulei and processing bodies (P- bodies) in the cytoplasm of mammalian cells. The organization of MCC domains in the yeast plasma membrane (Membrane compartment of Can1) was studied as well. It was shown that nonspecific interactions as the result of macromolecular crowding could be one of the main driving forces in formation and stabilization of these...
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U2 snRNA targeting to Cajal bodies
Roithová, Adriana ; Staněk, David (advisor) ; Mašek, Tomáš (referee)
In the cell we can find a lot of small noncoding RNAs, which are important for many processes. Among those RNAs are small nuclear RNA uridin rich, which with proteins create U snRNP.These particles play important role in pre-mRNA splicing. In this process are noncoding sequences (introns) removed and coding sequences (exons) are joined. It is catalyzed by spliceosome. The core of this spliceosome is created by U1, U2, U4, U5 and U6 snRNP. They are essential for this process. Some steps of U snRNP biogenesis proceed in nuclear structures called Cajal bodies (CB). In my thesis I focused on factors, which are important for targeting U snRNA into CB. I used U2 snRNA like a model. With the aid of microinjection of fluorescently labeled U2 snRNA mutants I found, that the Sm binding site on U2 snRNA is essential for targeting to CB. Knock down of Sm B/B'showed us, that Sm proteins are necessary for transport U2 snRNA to CB. Sm proteins are formed on U2 snRNA by SMN complex. Deletion of SMN binding site on U2 snRNA had the same inhibition effect. From these results we can see, that Sm proteins and SMN complex are important for U2 snRNA biogenesis espacially for targeting into CB. Key words: U snRNP, Cajal body, U snRNA, cell nucleus
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Study of the organization and dynamics of the membraneless cell compartments
Blažíková, Michaela ; Heřman, Petr (advisor) ; Cmarko, Dušan (referee) ; Hašek, Jiří (referee)
of Doctoral Thesis Title: Study of the organization and dynamics of the membraneless cell compartments Author: Michaela Blažíková Institute: Charles University in Prague, Faculty of Mathematics and Physics, Institute of Physics of Charles University Supervisor: Doc. RNDr. Petr Heřman, CSc., Charles University in Prague, Faculty of Mathematics and Physics, Institute of Physics of Charles University Abstract Eukaryotic cells contain many organelles and specific bodies. Beside the membrane delimited organelles such as nucleus, mitochondria or Golgi apparatus there are other structurally and functionally distinct membraneless structures in the cells. In this work we studied the self-organization processes, i.e. the processes that do not require specific interactions, of membraneless structures in nuclei, cytoplasm and plasma membrane of mammalian cells and yeast. The research was focused on the formation of nucleoli and Cajal bodies in mammalian cell nulei and processing bodies (P- bodies) in the cytoplasm of mammalian cells. The organization of MCC domains in the yeast plasma membrane (Membrane compartment of Can1) was studied as well. It was shown that nonspecific interactions as the result of macromolecular crowding could be one of the main driving forces in formation and stabilization of these...
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Functional analysis of hPrp8 mutations linked to retinitis pigmentosa.
Matějů, Daniel ; Cvačková, Zuzana (advisor) ; Král, Vlastimil (referee)
hPrp8 is an essential pre-mRNA splicing factor. This highly conserved protein is a component of the U5 small ribonucleoprotein particle (U5 snRNP), which constitutes one of the building blocks of the spliceosome. hPrp8 acts as a key regulator of spliceosome activation and interacts directly with U5 snRNA and with the regions of pre-mRNA that are involved in the transesterification reactions during splicing. Mutations in hPrp8 have been shown to cause an autosomal dominant form of retinitis pigmentosa (RP), an inherited disease leading to progressive degeneration of retina. In this study, we analyzed the effects of the RP-associated mutations on the function of hPrp8. Using BAC recombineering, we created mutant variants of hPrp8-GFP construct and we generated stable cell lines expressing the recombinant proteins. The mutant proteins were expressed and localized to the nucleus. However, one of the missense mutations affected the localization and stability of hPrp8. Further experiments suggested that RP-associated mutations affect the ability of hPrp8 to interact with other components of the U5 snRNP and with pre-mRNA. We further studied the biogenesis of U5 snRNP. We depleted hPrp8 by siRNA to interfere with U5 snRNP assembly and we observed that the incompletely assembled U5 snRNPs accumulate in...
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