Investigation of the global translational response to oxidative stress in the model archaeon reveals untranslated small RNAs with ribosome occupancy.

Oxidative stress induces a wide range of cellular damage, often causing disease and cell death. While many organisms are susceptible to the effects of oxidative stress, haloarchaea have adapted to be highly resistant. Several aspects of the haloarchaeal oxidative stress response have been characterized; however, little is known about the impacts of oxidative stress at the translation level. Using the model archaeon , we performed RNA-seq and ribosome profiling (Ribo-seq) to characterize the global translation landscape during oxidative stress. We identified 281 genes with differential translation efficiency (TE). Downregulated genes were enriched in ribosomal and translation proteins, in addition to peroxidases and genes involved in the TCA cycle. We also identified 42 small noncoding RNAs (sRNAs) with ribosome occupancy. Size distributions of ribosome footprints revealed distinct patterns for coding and noncoding genes, with 12 sRNAs matching the pattern of coding genes, and mass spectrometry confirming the presence of seven small proteins encoded by these sRNAs. However, the majority of sRNAs with ribosome occupancy had no evidence of coding potential. Of these ribosome-associated sRNAs, 12 had differential ribosome occupancy or TE during oxidative stress, suggesting that they may play a regulatory role during the oxidative stress response. Our findings on ribosomal regulation during oxidative stress, coupled with potential roles for ribosome-associated noncoding sRNAs and sRNA-derived small proteins in , revealed additional regulatory layers and underscored the multifaceted architecture of stress-responsive regulatory networks.IMPORTANCEArchaea are found in diverse environments, including as members of the human microbiome, and are known to play essential ecological roles in major geochemical cycles. The study of archaeal biology has expanded our understanding of the evolution of eukaryotes, uncovered novel biological systems, and revealed new opportunities for applications in biotechnology and bioremediation. Many archaeal systems, however, remain poorly characterized. Using as a model, we investigated the global translation landscape during oxidative stress. Our findings expand current knowledge of translational regulation in archaea and further illustrate the complexity of stress-responsive gene regulation.