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Role of the SMN complex in snRNP biogenesis
Hanušková, Klára ; Staněk, David (advisor) ; Šubrtová, Adriána (referee)
Small nuclear ribonucleoprotein particles (snRNPs) are an essential component of the spliceosome, a dynamic protein complex that mediates RNA splicing. These particles are composed of a single small nuclear RNA, from which they take their name, a ring of seven Sm or LSm proteins and other accessory proteins. All snRNPs undergo a complex maturation pathway, taking place in the nucleus and cytoplasm of the cell. One important factor in snRNP biogenesis is the SMN complex, which is composed of the SMN, Gemin2-8 and Unrip proteins. Its most important function is to mediate the assembly of the Sm core domain in the cytoplasm of the cell, with the help of the PRMT5 complex. Furthermore, the SMN complex is also involved in snRNA modification, import of the snRNP core, or assembly of the spliceosome itself. Despite more than two decades of research, some of the functions of the SMN complex and its particles have not been fully revealed, and thus it is not clear from the cell to what extent this complex influences not only snRNP biogenesis but also the entire spliceosome. The aim of this work, according to the available research, is to describe the SMN complex in more depth and to understand its functions in snRNP particle biogenesis. Key words: SMN complex, snRNP, snRNA, Sm protein, 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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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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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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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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