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Spliceosome assembly
Hausnerová, Viola ; Staněk, David (advisor) ; Chalupníková, Kateřina (referee)
Pre-mRNA splicing is a process in which introns are removed from eukaryotic transcripts and exons are ligated together. Splicing is catalyzed by spliceosome, a large ribonucleoprotein complex composed of five small nuclear RNAs and more than 100 additional proteins, which recognizes 5' splice site, branch point site and 3' splice site and performs two transesterification reactions to produce mRNA molecules. 5' splice site is recognized by U1 snRNP and U2 auxiliary factor (U2AF) is involved in branch point and 3' splice site recognition in the early splicing complex. There is some evidence of splice sites cooperation during intron recognition in vitro but little is known about the situation in vivo. Using Fluorescence resonance energy transfer (FRET) and RNA immunoprecipitation (RIP) methods, we have investigated the early stages of spliceosome assembly. We have employed splicing reporters based on -globin gene and MS2 stem loops to detect interactions of proteins on RNA molecule directly in the cell nucleus. Results of FRET indicate that intact 5' splice site is required for U2AF35 interaction with 3' splice site and that U1C recruitment to 5' splice site is partially limited upon 3' splice site mutation. We have also confirmed by RIP that U2 snRNP association with pre-mRNA molecule requires presence of 5'...
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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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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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The function of Slu7 protein in Saccharomyces cerevisiae pre-mRNA splicing
Ničová, Eva ; Půta, František (advisor) ; Vašicová, Pavla (referee)
Alternative splicing is one of the mechanisms how to regulate gene expression. Under different conditions, different mRNAs encoding proteins with different function, localization or stability can be made from one cellular transcript. The human hSlu7 protein affects the alternative splicing of some genes through alternative 3'splice site (3'SS) selection. Although it was thought that alternative splicing is absent from Saccharomyces cerevisiae, recent results argue against such conclusion. We therefore decided to characterize the function of the yeast Slu7 protein, which participates in the second step of splicing and is closely associated with the 3'SS selection. We focused on a highly conserved uncharacterized motif in the essential part of the Slu7 protein named the RED motif. Mutations in this motif caused second step splicing defects with some substrates and altered the alternative 3'SS usage ratio of some splicing constructs. Our results implicate a role for the RED motif in selecting proper 3'splice sites, especially the distal ones. Genetic interactions of slu7 mutations with PRP22 and PRP45 mutant alelles add to the intricate interaction network of splicing factors and suggest a possible role of Slu7p in facilitating the Prp22p association with the spliceosome.
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Molecular mechanism of quality control during snRNP biogenesis
Klimešová, Klára ; Staněk, David (advisor) ; Krásný, Libor (referee) ; Vomastek, Tomáš (referee)
The spliceosome is one of the largest and most dynamic molecular machines in the cell. The central part of the complex is formed by five small nuclear ribonucleoproteins (snRNPs) which are generated in a multi-step biogenesis pathway. Moreover, the snRNPs undergo extensive rearrangements during the splicing and require reassembly after every intron removal. Both de novo assembly and post-splicing recycling of snRNPs are guided and facilitated by specific chaperones. Here, I reveal molecular details of function of two snRNP chaperones, SART3 and TSSC4. While TSSC4 is a previously uncharacterized protein, SART3 has been described before as a U6 snRNP-specific factor which assists in association of U6 and U4 particles into di-snRNP, and is important for the U4/U6 snRNP recycling. However, the mechanism of its function has been unclear. Here, I provide an evidence that SART3 interacts with a post-splicing complex and propose that SART3 could promote its disassembly. Our data further suggest that SART3 binds U6 snRNP already within the post-splicing complex and thus participates in the whole recycling phase of U6 snRNP. Then, I show that TSSC4 is a novel U5 snRNP-specific chaperone which promotes an assembly of U5 and U4/U6 snRNPs into a splicing-competent tri-snRNP particle. We identified...
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Influence of transcription regulatory elemets on pre-mRNA splicing
Volek, Martin ; Staněk, David (advisor) ; Malík, Radek (referee)
In the process of pre-mRNA splicing introns are removed from pre-mRNA and exons are joined together. Current studies show, that about 95 % of genes, which contain more than two exons, can undergo alternative splicing. In this process some exons are included in or excluded from the final mRNA. Majority of pre-mRNA splicing take place co- transcriptionaly at this time RNA polymerase II is still attached to pre-mRNA. Alternative splicing is complex process that takes place in a close proximity of DNA and histones that might modulate alternative splicing decisions. Futher studies have validated fibronectin gene (FN1) and his alternative exons EDA and EDB (extra domain A and B) as suitably model for studying alternative splicing. Study using FN1 minigene reporter system, which is composed from EDA exon and two surrounding introns and exons, has proved that insertion of transcription enhancer SV40 infront of promotor, the level of EDA inclusion is decreased. So far, has not been prooved if this mechanism can function in real genome context and if distal transcription elements can influence alternative splicing. In this study, we have predicted transcription enhancer for FN1 gene by using The Ensemble Regulatory Build and FANTOM 5. The predicted transcription enhancer, is located 23,5 kbp upstream of TSS...
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Ribosomal protein Rpl22 regulates the splicing of its own transcripts
Nemčko, Filip ; Abrhámová, Kateřina (advisor) ; Müller-McNicoll, Michaela (referee)
Saccharomyces cerevisiae is an intron-poor organism with introns present in only 5% of its genes. The most prominent group of intron-containing genes are ribosomal protein (RP) genes. They are highly expressed and most of them are present as two paralogs. Parenteau et al. described the existence of intron- dependent intergenic regulatory circuits controlling expression ratios of RP paralogs. In this project, we did not confirm the regulation in 6 out of 7 tested regulatory circuits. We validated the regulation between RPL22 paralogs. We further showed that Rpl22 protein blocks the pre-mRNA splicing of both paralogs, with RPL22B paralog being more sensitive. Rpl22 protein binds to the stem-loop of RPL22B intron - disruption of the binding domain of Rpl22 proteins leads to loss of interaction. Moreover, the regulation seems to be working the same way in yeast Kluyveromyces lactis, which has only a single RPL22 copy. Overall, these results lead to better understanding of intergenic regulation, which adjusts the expression ratio between functionally different RPL22 paralogs. Key words introns, ribosomal protein genes, Rpl22, RPL22 paralogs, pre-mRNA splicing, Saccharomyces cerevisiae
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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
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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