Analysis and perturbation of C/D box sRNA-guided 2'-O-methylation patterns of Sulfolobus acidocaldarius ribosomal RNAs

• Applicant: Professor Dr. Lennart Randau
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; General Genetics and Functional Genome Biology
• Term: from 2016 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 315648833

In archaea and eukaryotes, ribonucleoprotein complexes exist containing small C/D box sno-like RNAs (sRNAs). These sRNAs contain two guide sequences that can exhibit base pair complementarity to target sites within ribosomal RNA (rRNA). The target nucleotides are subjected to 2'-O-ribose methylation, which aids in the folding and stabilization of nascent rRNA molecules. The genomes of hyperthermophilic archaea encode large numbers of C/D box sRNA genes, suggesting an increased necessity for rRNA stabilization at extreme growth temperatures. We have identified the complete sets C/D box sRNAs from seven archaea using Illumina RNA-Seq methodology. Their potential target sites were deduced from the guide sequences. These analyses revealed that C/D box sRNAs could acts as RNA chaperones that use their two guide regions to link target sites during rRNA folding. In our proposed research we plan to verify cellular 2'-O-ribose methylation patterns to be able to compare the targeting potential within the C/D box sRNA guides with experimentally observed modifications. Analyses will be performed in the genetically tractable hyperthermophilic archaeon Sulfolobus acidocaldarius, which allows us to perturb the C/D box sRNA-guide machinery. We aim to establish a method that combines the analysis of reverse transcriptase termination at 2'-O-ribose methylation sites (RTL-P) with RNA-Seq methodology. These global studies will allow us to detect the effects of individual C/D box sRNA gene deletions on the methylation patterns of ribosomal RNAs. We aim to delete C/D box sRNA genes for RNAs that (i) were shown to target the most conserved archaeal rRNA methylation sites and (ii) are proposed to contain linked guide sequences that could be used to coordinate rRNA folding. Finally, we will investigate if changes in the Sulfolobus acidocaldarius growth temperature affect stringency of C/D box sRNA/rRNA annealing, which should influence ribosome heterogeneity.

• DFG Programme: Research Grants