Characterization of SRP RNA circularization and Nudix hydrolase activity in the hyperthermophilic archaeon Sulfolobus acidocaldarius
Thermophilic archaea inhabit some of the most extreme environments on this planet. In adapting to their habitats, they have evolved numerous mechanisms to ensure the functionality of RNA molecules that are essential for key biological processes. This work describes the characterization of (i) the ci...
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|Summary:||Thermophilic archaea inhabit some of the most extreme environments on this planet. In adapting to their habitats, they have evolved numerous mechanisms to ensure the functionality of RNA molecules that are essential for key biological processes. This work describes the characterization of (i) the circularization of the universal signal recognition particle (SRP) RNA and (ii) the activities of a family of hydrolases suspected to play a role in non-canonical RNA decapping. The hyperthermophilic organism Thermoproteus tenax possesses a permuted SRP RNA gene whose transcript is processed by the tRNA splicing machinery into a circular SRP RNA proposed to possess elevated thermal stability due to its circular nature. To imitate a proposed genomic rearrangement event, a synthetic, permuted SRP RNA gene was implemented in the related thermophile Sulfolobus acidocaldarius, which contains a conventional SRP RNA gene. We show that S. acidocaldarius is capable of generating a circular SRP RNA from the synthetic gene variant, however swapping original and permuted gene variants was unsuccessful. Following the discovery of 5’-NAD-RNA in Archaea, this work investigates all four Nudix family hydrolases of S. acidocaldarius to uncover their RNA decapping potential. The recombinant proteins were produced and purified, but did not show activity against 5’-NAD-RNA. Instead, the protein SACI_RS00060, a putative ADPR-pyrophosphatase, revealed 5’-ADPR-RNA decapping activity in vitro and is suggested to play a role in the protection against thermal degradation of NAD(-RNA). In agreement, SACI_RS00060 also hydrolyzed free ADPR and was renamed Saci_NudT5. Analysis of the substrate preferences of the other Nudix proteins showed that SACI_RS00730 hydrolyzes the dinucleoside polyphosphate Ap4A and SACI_RS00575 exhibits strong preference for guanosine-based substrates. While deletions of the respective Nudix genes did not elicit a distinct phenotype under nutrient limitation or heat stress conditions, transcriptomic analyses of the Nudix deletion strains revealed a large variety of affected cellular processes, most likely due to effects on transcriptional regulators. Notably, the Nudix deletion transcriptomes resemble those of samples taken under heat stress and nutrient-limited conditions. The results of this work predict a connection of at least one Nudix hydrolase to heat stress response in S. acidocaldarius. In conclusion, this work provides first insights into specific substrate activities for each Nudix protein and lays the groundwork for the analysis of their impact on the metabolism of S. acidocaldarius.|
|Physical Description:||137 Pages|