Pan-archaeal analysis of C/D box sRNA biogenesis and methylation targets
Post-transcriptional modifications of RNA molecules occur in all three domains of life and influence RNA stability and functionality. The most numerous modifications are 2'-O-methylations at the ribose moiety and pseudouridylations. In archaea, modified bases are abundant in ribosomal RNAs (rRN...
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|Zusammenfassung:||Post-transcriptional modifications of RNA molecules occur in all three domains of life and influence RNA stability and functionality. The most numerous modifications are 2'-O-methylations at the ribose moiety and pseudouridylations. In archaea, modified bases are abundant in ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs). The introduction of both modifications is guided by small RNAs that are incorporated into ribonucleoprotein complexes (RNPs). 2'-O-methylations are guided by C/D box sRNAs in archaea. C/D box sRNAs are characterized by the conserved sequence elements boxC/C' (consensus sequence: RUGAUGA) and boxD/D' (consensus sequence: CUGA). Upon C/D box sRNA folding, both sequence elements base-pair (boxC with boxD and boxC' with boxD'), which results in the formation of two kink-turn motifs that are stabilized by binding of the protein L7Ae. The sequences between the two kink-turn elements show complementarity to the sequences of the target RNA and thereby serve as guide sequences that determine the sites of 2'-O-methylation. The modifications are introduced site-specifically at the nucleotide of the target RNA that is complementary to the fifth nucleotide upstream of the boxD/D' motif by the methyltransferase fibrillarin. Based on the guide sequences, C/D box sRNA targets of seven archaea were predicted and mapped onto the consensus structure of the 16S and 23S rRNA. Conserved methylation hotspots were observed in ancient core regions of the rRNAs that are important for ribosome integrity and functionality and that are not protected by ribosomal proteins. Therefore, the modifications might contribute to the folding, structural stabilization and function of the rRNAs. The biogenesis of archaeal C/D box sRNAs is largely unknown as independent promoters cannot be identified for the majority of the C/D box sRNA genes. The analysis of C/D box sRNA genes in six archaeal model organisms revealed diverse genetic contexts, providing opportunities for transcription without the necessity of an independent promoter. C/D box sRNA genes localize e.g. in the 5' or 3'-UTR of flanking protein-coding regions and polycistronic C/D box sRNA transcripts exist. Plasmid-based C/D box sRNA in vivo analyses were performed in Sulfolobus acidocaldarius in which C/D box sRNA genes variants with their native or random upstream and downstream sequences were used to identify C/D box sRNA stabilization and maturation requirements. The analyses revealed that the maturation of C/D box sRNAs occurs independently of the upstream and downstream sequences. The integrity of the k-turn is important for C/D box sRNA stability. Archaeal C/D box sRNAs exhibit a transcriptional plasticity and their maturation is suggested to include the action of unspecific exoribonucleases. Complete degradation might be prevented by co-transcriptional L7Ae binding or complete C/D box sRNP assembly. Circular forms of the C/D box sRNAs were identified in several hyperthermophilic archaea and the circularization reaction should protect RNAs from degradation. C/D box sRNA gene upstream and downstream sequences were shown not to be required for circularization but the responsible RNA ligase remains to be identified. Thus, this thesis provides insights into the transcription and maturation of archaeal C/D box sRNAs and highlights conserved 2'-O-methylation pattern in archaeal rRNAs.|