Small RNA-guided processes in the hyperthermophilic methanogen Methanopyrus kandleri
In this thesis, a combination of RNAseq, computational and biochemical methods was applied to analyze processes that use small RNAs (sRNAs) as guide molecules at extreme temperatures. Here, the hyperthermophilic archaeon Methanopyrus kandleri, which grows at temperatures of up to 110°C, was used as...
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|In this thesis, a combination of RNAseq, computational and biochemical methods was applied to analyze processes that use small RNAs (sRNAs) as guide molecules at extreme temperatures. Here, the hyperthermophilic archaeon Methanopyrus kandleri, which grows at temperatures of up to 110°C, was used as a model organism.
The genome of M. kandleri harbors two CRISPR-Cas systems that use CRISPR RNA (crRNA) as guide molecules to target foreign nucleic acids. RNAseq analysis revealed a high abundance and processing of crRNAs in M. kandleri that indicated that CRISPR-Cas systems are highly active at extreme temperatures. Furthermore, the crystal structure of the CRISPR-associated protein Csm3 was solved in collaboration with Prof. Dr. Elena Conti (MPI Martinsried). Csm3 was found to bind crRNAs and was shown to function as the crRNA-binding backbone protein in type III-A CRISPR-Cas interference complexes. A recently discovered nucleic acid-guided mechanism uses prokaryotic Argonaute (pAgo) proteins. In M. kandleri, a pAgo protein was found to be encoded within a potential operon of CRISPR-associated genes and the analysis of recombinant pAgo protein production revealed a high toxicity in Escherichia coli that might correlate with its potential defense function against plasmid DNA.
Methylation of rRNA is regulated by a different sRNA-guided mechanism that utilizes C/D box sRNAs to target a ribonucleoprotein complex to the rRNA methylation site. In M. kandleri, a record number of 126 C/D box sRNAs were detected by RNAseq analysis and indicate an increased potential for rRNA methylation reactions. Furthermore, most of the C/D box sRNAs were detected as circular molecules. Taken together, the circularization of C/D box sRNAs and the high requirement for rRNA methylation are suggested to be adaptations to the hyperthermophilic lifestyle of M. kandleri.
Finally, RNAseq analyses were used to identify tRNA precursors in M. kandleri that feature a unique C-to-U editing reaction of base 8. The occurrence of this editing event was used to deduce the order of tRNA processing steps in a non-compartmentalized cell, indicating that termini truncation precedes intron removal and editing.