RESUMEN
Facing rapid fluctuations in their natural environment, extremophiles, like the hyperthermophilic archaeon Pyrococcus furiosus, exhibit remarkable adaptability to extreme conditions. However, our understanding of their dynamic cellular responses remains limited. This study integrates RNA-sequencing and mass spectrometry data, thereby elucidating transcriptomic and proteomic responses to heat and cold shock stress in P. furiosus. Our results reveal rapid and dynamic changes in gene and protein expression following these stress responses. Heat shock triggers extensive transcriptome reprogramming, orchestrated by the transcriptional regulator Phr, targeting a broader gene repertoire than previously demonstrated. For heat shock signature genes, RNA levels swiftly return to baseline upon recovery, while protein levels remain persistently upregulated, reflecting a rapid but sustained response. Intriguingly, cold shock at 4°C elicits distinct short- and long-term responses at both RNA and protein levels. Cluster analysis identified gene sets with either congruent or contrasting trends in RNA and protein changes, representing well-separated arCOG groups tailored to their individual cellular responses. Particularly, upregulation of ribosomal proteins and significant enrichment of 5'-leadered sequences in cold-shock responsive genes suggest that translation regulation is important during cold shock adaption. Further investigating transcriptomic features, we reveal that thermal stress genes are equipped with basal sequence elements, such as strong promoter and poly(U)-terminators, facilitating a regulated response of the respective transcription units. Our study provides a comprehensive overview of the cellular response to temperature stress, advancing our understanding of stress response mechanisms in hyperthermophilic archaea and providing valuable insights into the molecular adaptations that facilitate life in extreme environments.IMPORTANCEExtreme environments provide unique challenges for life, and the study of extremophiles can shed light on the mechanisms of adaptation to such conditions. Pyrococcus furiosus, a hyperthermophilic archaeon, is a model organism for studying thermal stress response mechanisms. In this study, we used an integrated analysis of RNA-sequencing and mass spectrometry data to investigate the transcriptomic and proteomic responses of P. furiosus to heat and cold shock stress and recovery. Our results reveal the rapid and dynamic changes in gene and protein expression patterns associated with these stress responses, as well as the coordinated regulation of different gene sets in response to different stressors. These findings provide valuable insights into the molecular adaptations that facilitate life in extreme environments and advance our understanding of stress response mechanisms in hyperthermophilic archaea.
RESUMEN
RNA structural conformation and dynamics govern the functional properties of all RNA/RNP. Accordingly, defining changes of RNA structure and dynamics in various conditions may provide detailed insight into how RNA structural properties regulate the function of RNA/RNP. Traditional chemical footprinting analysis using chemical modifiers allows to sample the dynamics and conformation landscape of diverse RNA/RNP. However, many chemical modifiers are limited in their capacity to provide unbiased information reflecting the in vivo RNA/RNP structural landscape. In the recent years, the development of selective-2'-hydroxyl acylation analyzed by primer extension (SHAPE) methodology that uses powerful new chemical modifiers has significantly improved in vitro and in vivo structural probing of secondary and tertiary interactions of diverse RNA species at the single nucleotide level.Although the original discovery of Archaea as an independent domain of life is intimately linked to the technological development of RNA analysis, our understanding of in vivo RNA structural conformation and dynamics in this domain of life remains scarce.This protocol describes the in vivo use of SHAPE chemistry in two evolutionary divergent model Archaea, Sulfolobus acidocaldarius and Haloferax volcanii.