Investigation of the global translational response to oxidative stress in the model archaeon Haloferax volcanii reveals untranslated small RNAs with ribosome occupancy [Ribo-Seq]
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 many 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 that of coding genes, and mass spectrometry confirming the presence of seven small proteins originating from 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 during stress response. Overall design: We performed RNA-seq and Ribo-seq using wild-type H. volcanii cultures with and without oxidative stress treatment, using four biological replicates for each condition. We additionally performed RNA-seq on 3 sRNA knockout strains with oxidative stress treatment, using three biological replicates for each. Three biological replicates of wild-type, with and with stress treatment, were included as controls.