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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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Role of the ubiquitin-like protein, Hub1, in the pre-mRNA splicing regulation
Hubáčková, Tereza ; Abrhámová, Kateřina (advisor) ; Krchňáková, Zuzana (referee)
Splicing is a key step of eukaryotic gene expression and as well as other steps of this vital process, splicing has to be tightly regulated. Hub1 protein is a ubiquitin-like protein which noncovalently interacts with spliceosomal proteins Snu66 and Prp5 ATPase. According to the proposed model, low level of Hub1 protein stimulates ATPase activity of Prp5 helicase sufficiently for splicing of optimal splice sites, but not for splicing of suboptimal ones. Nevertheless, high level of Hub1 protein stimulates Prp5 ATPase sufficiently for splicing of both splice-site types. Excessive level of Hub1 protein may be harmful for the cell, because the immoderate splicing of suboptimal splice sites may produce aberrantly-spliced transcripts as a by-product. Hub1-induced negative feedback loop safeguards the cell from Hub1 protein hyperactivity by regulation of Prp5 ATPase level. Additionally, Hub1 protein regulates alternative splicing of Saccharomyces cerevisiae SRC1 gene and ensures appropriate balance of its products.
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Identification and characterization of proteins interacting with plant formins
Houšková, Anežka ; Cvrčková, Fatima (advisor) ; Abrhámová, Kateřina (referee)
Formins are evolutionarily conserved proteins participating in actin and microtubule organisation, affecting thus also intracellular transport, cell growth, morphogenesis and cell polarity. All formins contain FH2 domain, known to dimerize and act as a nucleator of actin. Angiosperms have two formin clades, Class I and Class II, which are distinguished by domain organisation. Based on knowledge from animal models and protein sequence homology, two groups of candidate membrane-associated formin interactors have been proposed in Arabidopsis (Cvrčková, 2013). First group of candidates consists of FYVE domain-containing proteins FAB1A (At4g33240) and FAB1B (At3g14270), the other contains proteins with BAR and SH3 domains AtSH3P1 (At1g31440), AtSH3P2 (At4g346600) and AtSH3P3 (At4g18060). Yeast two hybrid assay was used to examine protein interactions of selected proteins from both candidate groups (FAB1A, SH3P2 and SH3P3) with FH2 domains representing both plant formin clades. The same experimental setup was also used to test dimerization among FH2 domains of plant formins. Translational fusions of FH2 domains from Class I formins AtFH1 (At3g25500), AtFH5 (At5g54650) and Class II representatives AtFH13 (At5g58160) and AtFH14 (At1g31810) with the GAL4 activation domain have been co-expressed in yeast with GAL4...
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Gene expression regulation of ribosomal protein genes
Nemčko, Filip ; Abrhámová, Kateřina (advisor) ; Vopálenský, Václav (referee)
Saccharomyces cerevisiae cells produce 2000 ribosomes per minute under normal conditions. The expression of ribosomal proteins is massive - it takes 50% of RNA polymerase II transcription and 90% of pre-mRNA splicing in rapidly growing cells. Since cells need an equimolar amount of individual ribosomal proteins, the tight coregulation of gene expression is required. The transcription is a main target of regulation, however, it is inherently unable to set a stoichiometric balance of ribosomal proteins. Various types of post-transcriptional regulation deal with fluctuations of individual ribosomal proteins and fine-tune their expression. Intron-dependent regulation appears to by predominant among ribosomal protein genes. Besides balancing their expression, presence of introns provides a rapid global regulation (repression) of ribosomal protein genes in response to environmental stress. KEY WORDS ribosomal protein genes, RPG, ribosomal protein, gene expression regulation, coregulation, Saccharomyces cerevisiae
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Mer1 and Nam8 in splicing regulation
Marková, Michaela ; Abrhámová, Kateřina (advisor) ; Stejskalová, Eva (referee)
By the mutual cooperation, Mer1 nad Nam8 proteins activate pre-mRNA splicing of four specific genes: AMA1, MER2, MER3 and SPO22, that are required for meiotic recombination and nuclear division. Expression of these genes does not change during the cell cycle, nevertheless, the efficient splicing of their pre-mRNA occures only during meiosis because Mer1 protein, which facilitates their splicing, is expressed only in meiotic cells. All these pre-mRNAs contain nonconsensual 5'splice site (5'ss) which is less recognizable for the spliceosomal subunit U1 snRNP in comparison with the consensual sequence. There is an enhancer area near to 5'ss on the pre-mRNA of this genes that serves as a binding site for Mer1p which makes recruitment of U1 snRNP more efficient. Mer1p cooperate via other proteins with Nam8p. Protein Nam8, a part of U1 sn RNP, is bound on pre-mRNA downstream from 5'ss close to the enhancer area. Mer1p in cooparation with Nam8p facilitates spliceosome assembly on the nonconsensual 5'ss and subsequently pre-mRNA splicing.
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The role of Prp45p in mRNA expression and maturation
Abrhámová, Kateřina ; Půta, František (advisor) ; Pichová, Alena (referee) ; Janderová, Blanka (referee)
Prp45p of Saccharomyces cerevisiae and Snw1p of Schizosaccharomyces pombe are essential proteins, which share extensive homology with the mammalian transcription regulator and splicing factor SNW/SKIP. We have analyzed the essential function of these proteins in both yeasts and found a mutation (prp45(1-169)) that exhibited temperature sensitivity. The mutant strain harboring the corresponding chromosomal deletion shows temperature sensitive phenotype and hypersensitivity to cycloheximide, hydroxyurea, calcofluor white, and to microtubule inhibitors. At 30řC, the cells are often elongated, deformed, and larger than wt. After synchronization prp45(1-169) cells stop their growth with 2N DNA content at 37řC. We found that the temperature sensitivity is not overcome and the hypersensitivity to microtubule destabilizing drugs is only partially suppressed by the excision of intron from TUB1 gene. This distinguishes prp45(1-169) from those splicing factor's mutants that cause tubulin-dependent G2/M arrest, which can be relieved by the expression of intronless tub1. We performed analysis of splicing in vitro and found that splicing of optimal substrates is not impaired. We also compared the content and stability of RNA in wt-cells and in prp45(1-169) cells at variol temperatures using microarrays. The...
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