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Comparative analysis of RNA binding properties of Rpl22 proteins
Gredová, Alexandra ; Abrhámová, Kateřina (advisor) ; Vopálenský, Václav (referee)
After the whole genome duplication event, Saccharomyces cerevisiae lost 90 % of its paralogs. 59 ribosomal protein genes (RPG) retained a duplicated form. The cell has to balance expression of RPGs as a part of adaptation to changing conditions, thus ensuring the production of the right ratio of ribosomal proteins (RP). In addition, RPGs contain 1/3 of all introns found in the S. cerevisiae genome. Rpl22A/B are part of the large ribosomal subunit where they contact 25S rRNA. Deletion of these RPGs results in 2X slower growth in comparison with WT cells. Within their extraribosomal function Rpl22A/B are able to intragenically and intergenically regulate their expression. Binding of Rpl22A/B to the intronic part of pre-mRNA of RPL22A or RPL22B results in splicing inhibition, which is stronger in the case of the RPL22B intron (RPL22Bi). However, the exact mechanism is not known. We know that Rpl22s from various organisms bind a hairpin structure that can be found in 25S rRNA also. Since the predicted structure of RPL22Bi does not contain this binding motif, it rises a question of whether the RNA binding surface of Rpl22A/B is different or more extensive than the one with which Rpl22 contacts 25S rRNA. We compared the ability of Rpl22 from different organisms to complement the functions of Rpl22A/B. These...
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Analysis of co-transcriptional spliceosome assembly on RPL22B
Hájková, Karolína ; Abrhámová, Kateřina (advisor) ; Cvačková, Zuzana (referee)
In eukaryotic organisms, the process of splicing is used to remove non-coding sequences - introns - from transcripts to form mRNA, and together with transcription, it also allows to regulate the amount of the resulting RNA that will be used further in translation. The protein of interest in our study is Rpl22, which binds the intron of its own transcript and its paralog and can inhibit their splicing. This thesis examines the mechanism by which the inhibition occurs and provides new information about the regulation of RPL22 transcript expression via its intron. Using co-transcriptional spliceosome assembly analysis on the RPL22B transcript, we were able to observe that the recognition of splice sites by the U1 snRNP, Msl5 and Mud2, is not affected by the inhibition. Thus, the inhibition of RPL22B splicing will occur later during spliceosome assembly or during spliceosome activation, before the first step of splicing takes place.
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A role of the 5' cap in Sm-class snRNA biogenesis
Petržílková, Hana ; Staněk, David (advisor) ; Abrhámová, Kateřina (referee)
Small nuclear RNAs (snRNAs) are the core component of the spliceosome which catalyzes pre-mRNA splicing. They undergo a complex biogenesis process which includes snRNA modifications and their assembly into ribonucleoprotein splicing particles snRNPs. The snRNA biogenesis pathway also includes several quality control steps, which block defective snRNPs from entering the spliceosome. One of the important feature involved in Sm-class snRNA quality control is the 5' trimethylguanosine cap (TMG). The capping of Sm-class snRNAs is also connected to the modifications of the first transcribed nucleotide, adenosine, which gets co-transcriptionally 2'-O-methylated and N6-methylated. However, the N6-methylation is later removed by FTO demethylase. Here I shed more light on functional relevance of the various modifications of snRNA 5' cap. I show that the N6-demethylation of the cap is important for normal metabolism of snRNAs, especially U2 snRNA. Next, I provide evidence that the 5' cap plays a role in quality control of 5' truncated snRNAs. Our data suggest that the truncated snRNAs accumulate immature monomethylated caps and are bound by a specific cap binding complex IFIT1/2/3. I propose that the 5' truncated snRNAs are partially stalled in early stages of snRNA biogenesis and are targeted by quality...
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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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The role of coilin in snRNP quality control
Kuzmenko, Darya ; Staněk, David (advisor) ; Abrhámová, Kateřina (referee)
Mammalian genes are transcribed as precursors - pre-mRNA. They contain coding sequences (exons) and non-coding sequences (introns). Splicing, a process of cutting out introns and joining exons to generate mature mRNA, is carried out by a spliceosome. The spliceosome consists of five small nuclear ribonucleoprotein (snRNP) particles and numerous associated proteins. Its assembly is a complex process involving a specific nuclear sub-compartment, the Cajal body (CB). Here, we investigate function of the CB scaffold protein, coilin, in snRNP quality control in HeLa cells. Sequestration of immature snRNP in coilin-deficient cells is analysed by fluorescence in situ hybridisation. We show that without coilin the cells are unable to sequester them. Next, we provide evidence that absence of coilin does not sensitise HeLa cells for perturbation in snRNP maturation in terms of cell proliferation. Moreover, coilin deficiency does not result in significant changes in U4, U5 or U6 snRNA steady state levels. Therefore, coilin, and, in this way, Cajal bodies do not become essential under the conditions of strained snRNP biogenesis.
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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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Effect of SR-like proteins in maturation and transfer of mRNA to the cytoplasm
Hájková, Karolína ; Abrhámová, Kateřina (advisor) ; Staněk, David (referee)
Metazoan SR proteins is a group of proteins involved in splicing. They are RNA-biding proteins characterized by the presence of serine and arginine-rich domain. The yeast, Saccharomyces cerevisiae, encodes genes for proteins that are similar in amino acid composition and structure to the group of SR proteins and are therefore called SR-like proteins. There are three proteins with RS domain in yeast: Npl3, Gbp2 and Hrb1. These proteins have been shown to have many functions, including, in addition to splicing, quality control of spliced mRNAs and their export from the nucleus to the cytoplasm. This Bachelor's thesis will deal with these three yeast proteins and their role in splicing, export and degradation.
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Cryptococcus neoformans virulence factors
Bauer, Martin ; Kuthan, Martin (advisor) ; Abrhámová, Kateřina (referee)
Cryptococcus neoformans is an opportunistic pathogenic yeast causing around 600 000 deaths annually. Its ability to cause a chronic infection is given by multiple virulence factors, which include the ability to grow in high temperature, polysaccharide capsule, oxidative stress tolerance and the expression of surface proteins. Unusual and resistant titan cells, which develop through the process of titanization, and the process of phenotypic switching can also be included. Despite intensive research, these virulence factors are yet to be fully described. Inducing factors of titan cells and participating signalling pathways are known. However, a complete model of titanization does not yet exist. So far, the mechanism of phenotypic switching is also unknown. In this work the current knowledge of virulence factors of C. neoformans is presented and summarised.
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Phenotypic switching and cell differentiation in yeast Cryptococcus neoformans
Bauer, Martin ; Kuthan, Martin (advisor) ; Abrhámová, Kateřina (referee)
Cryptococcus neoformans is an opportunistic pathogenic yeast causing around 600 000 deaths annually. Its ability to cause a chronic infection is given by the emergence of different morfotypes. These morphotypes differ in cell structures and mechanisms (virulence factors) which have an influence on the resistance to stress factors encountered in the host. This work first describes molecular mechanisms of formation of these virulence factors. Next, it presents morphotypes occurring during infection and the hypovirulent pseudohyphal morphotype. However, this morphotype is interesting because of a modification in the signalisation leading to its manifestation. Finally, described signalling pathways present possible ways of regulating the virulence factors, and so the manifestation of different morphotypes. Understanding these signalling pathways could ultimately lead to improving the development of new drugs, given that Cryptococcus neoformans is highly resistant to the existing ones. Keywords: Cryptococcus neoformans, phenotypic switching, titan cells, cell differentiation, virulence, Vad1, Rim101, Usv101, RAM
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