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RNA directed DNA methylation in Arabidopsis thaliana
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The differential transcriptional activity of the genome is provided by epigenetic modifications, which include DNA methylation, alteration of histone N-terminal amino acids and changes in histone variants. RNA interference is a regulatory process, in which transcriptional or post-transcriptional silencing of exogenous or endogenous sequences is mediated by the action of small RNAs derived from these sequences. The 24-nucleotide siRNAs, forming a fraction of small RNAs, direct de novo DNA methylation and participate in the maintenance of DNA methylation (RNA-directed DNA methylation; RdDM), which facilitates transcriptional silencing of heterochromatin and transposable elements representing a large part of plant genomes. The presence of two RNA polymerases involved in this pathway is characteristic for flowering plants, which were discovered for the first time in the genome of Arabidopsis thaliana, which has also become the main plant model for the study of RdDM. Polymerase IV transcribes siRNA precursors; siRNAs are subsequently associated with AGO4 proteins and guide methylation enzymes to the target sequences via complementarity with polymerase V transcripts.
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Study of the mechanism of posttranscriptional and transcriptional transgene silencing in tobacco BY-2 cell line
Čermák, Vojtěch ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The RNA interference is a mechanism, which allows cells to regulate their genes functions, to establish and maintain heterochromatin and to defend them against invasive nucleic acids. In plants, RNA interference is initiated by double-stranded RNA, which is processed by Dicer into small RNAs, usually 20-24nt long. These small RNAs form a complex with Argonaut protein that participates in different processes based on sequence complementarity. This complex can guide mRNA cleavage, translation blocking and chromatin modifications, resulting either into posttranscriptional silencing (by preventing translation of already existing mRNA, PTGS) or transcriptional silencing (by preventing transcription of mRNA, TGS). The first step of this thesis was to establish different ways of triggering PTGS and to evaluate their functionality and efficiency. The next step was a preparation of a system which would allow to study the transition from posttrancriptional to transcriptional silencing. These so called "indicator lines" should allow to observe the timing and dynamics of this process by utilizing fluorescent proteins. This system is also going to enable to evaluate, how different factors are involved in this process - one of the factors is RNA-dependent RNA polymerase 6 (RDR6) which plays an essential role in...
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RNA directed DNA methylation in Arabidopsis thaliana
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The differential transcriptional activity of the genome is provided by epigenetic modifications, which include DNA methylation, alteration of histone N-terminal amino acids and changes in histone variants. RNA interference is a regulatory process, in which transcriptional or post-transcriptional silencing of exogenous or endogenous sequences is mediated by the action of small RNAs derived from these sequences. The 24-nucleotide siRNAs, forming a fraction of small RNAs, direct de novo DNA methylation and participate in the maintenance of DNA methylation (RNA-directed DNA methylation; RdDM), which facilitates transcriptional silencing of heterochromatin and transposable elements representing a large part of plant genomes. The presence of two RNA polymerases involved in this pathway is characteristic for flowering plants, which were discovered for the first time in the genome of Arabidopsis thaliana, which has also become the main plant model for the study of RdDM. Polymerase IV transcribes siRNA precursors; siRNAs are subsequently associated with AGO4 proteins and guide methylation enzymes to the target sequences via complementarity with polymerase V transcripts.
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Study of the mechanism of posttranscriptional and transcriptional transgene silencing in tobacco BY-2 cell line
Čermák, Vojtěch ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The RNA interference is a mechanism, which allows cells to regulate their genes functions, to establish and maintain heterochromatin and to defend them against invasive nucleic acids. In plants, RNA interference is initiated by double-stranded RNA, which is processed by Dicer into small RNAs, usually 20-24nt long. These small RNAs form a complex with Argonaut protein that participates in different processes based on sequence complementarity. This complex can guide mRNA cleavage, translation blocking and chromatin modifications, resulting either into posttranscriptional silencing (by preventing translation of already existing mRNA, PTGS) or transcriptional silencing (by preventing transcription of mRNA, TGS). The first step of this thesis was to establish different ways of triggering PTGS and to evaluate their functionality and efficiency. The next step was a preparation of a system which would allow to study the transition from posttrancriptional to transcriptional silencing. These so called "indicator lines" should allow to observe the timing and dynamics of this process by utilizing fluorescent proteins. This system is also going to enable to evaluate, how different factors are involved in this process - one of the factors is RNA-dependent RNA polymerase 6 (RDR6) which plays an essential role in...
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