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Functional analysis of the TSSC4 chaperone during snRNP formation
Vojáčková, Jitka ; Staněk, David (advisor) ; Vaňková Hausnerová, Viola (referee)
Splicing is a process, during which non-coding sequences (introns) are cleaved out of pre-mRNA, and exons are ligated. This whole process is catalysed by a multi-megadalton splicing complex, composed of five small nuclear ribonucleoprotein particles (shortly snRNPs), which each contains its own small nuclear RNA molecule and specific set of proteins. During the biogenesis of snRNPs, U4 and U6 snRNPs are assembled to form the di-snRNP, which further associates with U5 snRNP and gives rise to tri-snRNP. With the help of mass spectrometry, we have found previously uncharacterized protein interacting with U5 snRNP, called TSSC4. By immunoprecipitation, I confirmed TSSC4 as a U5 snRNP specific protein and identified the region of TSSC4 responsible for interaction with U5 snRNP. I also showed that TSSC4 interacts with PRPF19, a component of complex driving the catalytic activation of the spliceosome and that this interaction is U5 snRNP-independent. Knockdown of TSSC4 in HeLa cells results in accumulation of di-snRNAs and U5 snRNP in Cajal bodies, nuclear compartments involved in snRNP biogenesis. Similar phenotype was previously observed upon inhibition of tri-snRNP assembly. To analyse the importance of TSSC4 for tri-snRNP assembly, I separated individual snRNPs by glycerol gradient ultracentrifugation...
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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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Functional analysis of the TSSC4 chaperone during snRNP formation
Vojáčková, Jitka ; Staněk, David (advisor) ; Vaňková Hausnerová, Viola (referee)
Splicing is a process, during which non-coding sequences (introns) are cleaved out of pre-mRNA, and exons are ligated. This whole process is catalysed by a multi-megadalton splicing complex, composed of five small nuclear ribonucleoprotein particles (shortly snRNPs), which each contains its own small nuclear RNA molecule and specific set of proteins. During the biogenesis of snRNPs, U4 and U6 snRNPs are assembled to form the di-snRNP, which further associates with U5 snRNP and gives rise to tri-snRNP. With the help of mass spectrometry, we have found previously uncharacterized protein interacting with U5 snRNP, called TSSC4. By immunoprecipitation, I confirmed TSSC4 as a U5 snRNP specific protein and identified the region of TSSC4 responsible for interaction with U5 snRNP. I also showed that TSSC4 interacts with PRPF19, a component of complex driving the catalytic activation of the spliceosome and that this interaction is U5 snRNP-independent. Knockdown of TSSC4 in HeLa cells results in accumulation of di-snRNAs and U5 snRNP in Cajal bodies, nuclear compartments involved in snRNP biogenesis. Similar phenotype was previously observed upon inhibition of tri-snRNP assembly. To analyse the importance of TSSC4 for tri-snRNP assembly, I separated individual snRNPs by glycerol gradient ultracentrifugation...
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Formation of splicing machinery in the context of the cell nucleus
Stejskalová, Eva ; Staněk, David (advisor) ; Vanáčová, Štěpánka (referee) ; Malínský, Jan (referee)
Most of the protein coding genes of higher eukaryotes contain introns which have to be removed from primary transcripts to make mRNA which can be used as a template for protein synthesis. This crucial step in the pre-mRNA processing is carried out by the spliceosome, a complex ribonucleoprotein machine formed from small ribonucleoprotein particles (snRNPs). snRNPs biogenesis is a complex process composed of several steps which take place in both the cytoplasm and the nucleus. Spliceosome assembly is highly dynamic and tightly regulated and pre-mRNA splicing depends not only on the sequence of the pre-mRNA itself but also on the nuclear context, such as the chromatin modifications. How do cells regulate where and when the spliceosome would be assembled? What determines which introns will be spliced? These are fundamental, yet unanswered, biological questions. In this work we analyzed the formation of splicing machinery in the context of the cell nucleus from several different points of view. First, we investigated the unexpected connection between splicing factor U1-70K and the survival of motor neurons (SMN) complex which is a major player in the snRNP biogenesis pathway. We revealed that U1-70K interacts with the SMN complex and that this interaction is crucial for the stability of nuclear gems, small...
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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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