The hammerhead ribozyme structure brought in line.
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Base Sequence↗
Biomedical subjects
Christian Hammann
Publications and source records attributed to Christian Hammann.
Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi.
We have identified a DNA methyltransferase of the Dnmt2 family in Dictyostelium that was denominated DnmA. Expression of the dnmA gene is downregulated during the developmental cycle. Overall DNA methylation in Dictyostelium is approximately 0.2% of the cytosine residues, which indicates its restriction to a limited set of genomic loci. Bisulfite sequencing of specific sites revealed that DnmA is responsible for methylation of mostly asymmetric C-residues in the retrotransposons DIRS-1 and Skipper. Disruption of the gene resulted in a loss of methylation and in increased transcription and mobilization of Skipper. Skipper transcription was also upregulated in strains that had genes encoding components of the RNA interference pathway disrupted. In contrast, DIRS-1 expression was not affected by a loss of DnmA but was strongly increased in strains that had the RNA-directed RNA polymerase gene rrpC disrupted. A large number of siRNAs were found that corresponded to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA modification. No siRNAs corresponding to the standard Skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene silencing in Dictyostelium but that different, partially overlapping mechanisms control transposon silencing.
Amino Acid Sequence↗
Functional hammerhead ribozymes naturally encoded in the genome of Arabidopsis thaliana.
The hammerhead ribozyme (HHRz) is an autocatalytic RNA motif found in subviral plant pathogens and transcripts of repetitive DNA sequences in animals. Here, we report the discovery and characterization of unique HHRzs encoded in a plant genome. Two novel sequences were identified on chromosome IV of Arabidopsis thaliana in a database search, which took into account recently defined structural requirements. The HHRzs are expressed in several tissues and coexist in vivo as both cleaved and noncleaved species. In vitro, both sequences cleave efficiently at physiological Mg(2+) concentrations, indicative of functional loop-loop interactions. Kinetic analysis of loop nucleotide variants was used to determine a three-dimensional model of these tertiary interactions. Based on these results, on the lack of infectivity of hammerhead-carrying viroids in Arabidopsis, and on extensive sequence comparisons, we propose that the ribozyme sequences did not invade this plant by horizontal transfer but have evolved independently to perform a specific, yet unidentified, biological function.
Arabidopsis↗
A database search for double-strand containing RNAs in Dictyostelium discoideum.
In eukaryotic cells, double-stranded RNA is degraded to 21mers and triggers RNA interference. Using a pattern description language, we have searched the EMBL database for sequences with the potential to form double strands in cis in Dictyostelium discoideum. No extended inverted repeats were found in mRNAs. However, the antisense direction of some mRNAs encoding regulatory or developmentally regulated proteins showed the ability to form double-stranded regions. In EST archives, we found potential double strands derived from a few genes, but these transcripts are not continuously encoded in the genome. Most likely, they represent hybrid molecules of sense and antisense RNAs.
Animals↗
Folding and activity of the hammerhead ribozyme.
The hammerhead is the smallest of the nucleolytic ribozymes, that undergo backbone cleavage by a transesterification reaction in the presence of magnesium ions. The RNA is induced to fold into its active conformation by the binding of metal ions in two stages. These generate domain 2, the scaffold on which the ribozyme is built, and domain 1, the active centre of the ribozyme. Further local structural rearrangement during the activation of the ribozyme is suggested by a number of crystal structures. The 10(5)-fold rate enhancement is probably brought about by a combination of metal-ion participation and stereochemical factors in the environment of the folded RNA structure.
Base Sequence↗