Investigation of the global translational response to oxidative stress in the model archaeon Haloferax volcanii 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 Haloferax volcanii , 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 observed upregulated TE for several transporters and membrane-bound proteases, highlighting the importance of membrane dynamics during oxidative stress. We additionally 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 originating from 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. In combination with the evidence of regulation at the translation level during oxidative stress, this demonstrates the complexity of gene regulation in response to stress.