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Reactive modifications of RNA for bioconjugations with proteins and new enzymatic methods for the synthesis of base-modified RNA
Brunderová, Mária ; Hocek, Michal (advisor) ; Míšek, Jiří (referee) ; Vaňáčová, Štěpánka (referee)
This doctoral thesis focuses on the enzymatic synthesis of base-modified RNA probes with diverse functional groups, including reactive cross-linking, hydrophobic and fluorescent moieties, or affinity tags. The construction of nucleobase-modified oligonucleotides is accomplished either through conventional in vitro transcription with T7 RNA polymerase or by an innovative approach leveraging engineered mutant DNA polymerases and primer extension reaction (PEX). In the first section of the thesis, a novel ribonucleoside triphosphate building block with reactive chloroacetamide functionality was synthesised using an aqueous Pd-catalysed Sonogashira cross- coupling reaction, directly applied on iodinated nucleotide. The chloroacetamide modified triphosphate was then tested as a putative substrate for T7 RNA polymerase in in vitro transcription reaction, aiming to construct RNA probes with one or multiple reactive groups. The selectivity of chloroacetamide- modified RNA for thiol-, or cysteine-, and histidine-containing (bio)molecules was demonstrated by model bioconjugation reactions and cross-linking experiments with three RNA-binding proteins of diverse structures and functions. The efficient formation of RNA-protein covalent adducts was confirmed by western blot or gel, and mass spectrometry analyses...
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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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Long Non-Coding RNAs in Oocyte-to-Embryo Transition
Ganesh, Sravya ; Svoboda, Petr (advisor) ; Vanáčová, Štěpánka (referee) ; Shkumatava, Alena (referee)
(English) Oocyte-to-embryo transition (OET) is one of the most complex developmental events, during which a differentiated oocyte gives rise to a totipotent zygote. During OET a transcriptionally silent oocyte undergoes massive reprogramming of gene expression, which transforms it into a transcriptionally active zygote. Although numerous studies have contributed to understanding the mechanism of OET, many genes involved in OET are yet to be identified. A whole new level of possible regulation of OET came with the discovery of long non-coding RNAs (lncRNA). LncRNAs are pol II transcripts longer than 200 nucleotides, that are typically spliced and polyadenylated but do not encode proteins. While lncRNAs have been studied in many model systems including embryonic stem cells, their expression in oocytes and early embryos and contribution to OET were largely unexplored at the beginning of this project. In my PhD project, I aimed to identify, annotate, and analyze lncRNAs expressed during OET. First, using RNA-Seq, 1600 highly reliable lncRNAs were identified and annotated in mouse oocytes and early embryos. Majority of lncRNAs were novel with expression exclusively at OET stages. A significant fraction of these lncRNAs was found associated with LTR retrotransposons, contributing to their novelty and...
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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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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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Chromosomes of parasitic protist Trichomonas vaginalis
Zubáčová, Zuzana ; Tachezy, Jan (advisor) ; Alsmark, Cecília (referee) ; Vanáčová, Štěpánka (referee)
Charles University in Prague Faculty of Science Programme of study: Parasitology Abstract of Ph.D. thesis Chromosomes of parasitic protist Trichomonas vaginalis Zuzana Zubáčová, MSc. Supervisor: Prof. RNDr. Jan Tachezy, Ph.D. Praha, 2011 Abstract Genome sequencing and associated proteome projects has revolutionized research in the field of molecular parasitology. Progress in sequencing of parasite as well as free-living species enables comparative and phylogenetic studies and provides important data sets for understanding of basic biology and identification of new drug targets. The genome sequencing of Trichomonas vaginalis revealed surprisingly large genome size of this organism (~160 Mb) that resulted from expansion of various repetitive elements, specific gene families and large scale genome duplication. I studied genome sizes of other nine selected species from Trichomonadea group to find whether other trichomonads have undergone similar genome expansion. The measurement of nuclear DNA content by flow cytometry revealed relatively large DNA content in all tested species although within a broad range (86-177 Mb). The largest genomes were observed in the Trichomonas and Tritrichomonas genera while Tetratrichomonas contains the smallest genome. The genome sizes correlated with the cell volume however no...
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To cap or not to cap? Eukaryotic translation initiation with a special interest in human opportunistic pathogen C. albicans
Feketová, Zuzana ; Pospíšek, Martin (advisor) ; Půta, František (referee) ; Vanáčová, Štěpánka (referee)
Candida albicans belongs to serious human opportunistic pathogens, causing severe health complications to immunocompromised patients. To my best knowledge, it is the only organism that survives with unmethylated cap structures found on the 5'ends of mRNA molecules. Using functional assay, I demonstrated that orf19.7626 codes for C. albicans translation initiation factor 4E (Ca4E). We couldn't prove our hypothesis, that Ca4E could be responsible for the unmethylated cap recognition in our model organism S. cerevisiae. Candida sp. possesses also another rather unusual feature - ambiguous CUG codon. In most of the cases, CUG is decoded as a serine, but sometimes also as a leucine. This gives rise to a so called "statistical proteome". One CUG codon is also part of the mRNA coding for Ca4E protein, therefore two versions of Ca4E-Ca4ELeu and Ca4ESer -might occur in C. albicans simultaneously. Both of them are able to rescue deletion of S. cerevisiae eIF4E gene, but they confer temperature sensitivity to the heterologous host. This phenotype is more pronounced with the Ca4ELeu version. We observed milder temperature sensitive phenotype after co-expression of Ca4E together with C. albicans eIF4G (Ca4G). Conformational coupling between eIF4E and eIF4G leads to enhanced affinity of eIF4E to the cap...
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