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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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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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Molecular genetic examinations in clinically defined group of patients with syndromic sight and hearing impairment in rare genetic disorders associated with deafblindness in the CR and SR
Čopíková, Jana ; Kremlíková Pourová, Radka (advisor) ; Astl, Jaromír (referee) ; Baxová, Alice (referee)
Deafblindness is a combined impairment of vision and hearing with an incidence of about 1: 8000 children and 1: 5500 adults. The most common genetic causes are the Stickler (STL) and Usher (USH) syndromes. The main goal of this work is to provide an up-to-date overview of STL and USH in the Czech and Slovak Republic (CR and SR), to determine the correlations between the genotype and phenotype in our population and the associated diagnostic criteria. Using sequencing and MLPA we examined 45 patients from 28 families for suspected STL. We found potentially causal variants of STL genes in 39 patients from 22 families. Fifteen different COL2A1 variants (8 being novel) were found in 28 patients from 18 families and 4 novel COL11A1 variants were found in 11 patients from 4 families. We identified the cause of the disease in 79 % of the families. The USH study involved 30 patients from 27 families. The most frequent cause was USH2A pathogenic variants, i.e. 19 variants in 14 families, 9 being novel. Less common were pathogenic variants in MYO7A (6 variants in 3 families, 5 being novel), USH1C and CDH23 (3 variants, 2 being novel, in 2 families both) genes. In 2 families, compound heterozygosity was found for variants in two different USH genes. The deafblindness etiology was clarified for 24 patients from...
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The role of pre-mRNA splicing in human hereditary diseases
Malinová, Anna
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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Molecular genetic analysis of patients with Usher syndrome
Průšová, Kateřina ; Ďuďáková, Ľubica (advisor) ; Kousal, Bohdan (referee)
The work focuses on molecular genetic testing of patients with Usher syndrome to confirm the diagnosis, to determine the causal cause of the disease and describe new mutations causing Usher syndrome in Czech patients. Usher syndrome is a clinically and genetically heterogeneous disease that is the most common cause of hereditary deafblindness. Based on responsible genes and disease onset is classified into three clinical subtypes. Given the fact that there is currently no specific treatment, there is a need to understand the pathophysiology of this disease and to broaden the spectrum of causal mutations. The theoretical part of the thesis deals with the anatomy of the eye, especially the structure of the retina. Attention is also paid to retinal diseases, such as the progressive loss of vision characteristic for retinitis pigmentosa (RP). RP may occur either as an isolated disorder or also affecting other organs, so-called syndromic RP. Classic syndromic RP includes Usher's syndrome, which the work mainly deals with. The theoretical part of the thesis describes mainly the mechanism of the disease, the functions of individual Usher proteins and the genes that encode these proteins. The haplotype analysis has been previously done for the most common mutations causing Usher's syndrome in Europe Based...
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The role of pre-mRNA splicing in human hereditary diseases
Malinová, Anna
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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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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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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