Transcriptional Landscape and Regulatory Roles of Small Noncoding RNAs in the Oxidative Stress Response of the Haloarchaeon Haloferax volcanii

We demonstrate here the transcriptional landscape and functional roles of sRNAs specifically in the regulation of the oxidative stress response of the model haloarchaeon Haloferax volcanii. We sequenced 5 biological replicates of H. volcanii strain H53 under no challenge and oxidative stress (H2O2) conditions at mid-exponential phase (OD 0.4). Thousands of sRNAs, both intergenic and antisense, were discovered using strand-specific sRNA-seq, comprising around 30% of the transcriptome during non-challenged and oxidative stress conditions. Antisense sRNAs were found to overlap both 5’ and 3’ UTRs of mRNAs revealing a hybrid system between Eukarya (3’ UTR) and Bacteria (5’ UTR) sRNA-silencing systems, as well as targeting the coding sequence (CDS) of mRNAs, a unique system to Archaea. We identified hundreds of differentially expressed sRNAs in response to hydrogen peroxide induced oxidative stress in H. volcanii. A majority of these sRNAs are lowly expressed compared to mRNAs. The sRNAs could be classified in two populations based on expression patterns: those that are up-regulated and those that are down-regulated during oxidative stress. Targets of antisense sRNAs decreased in expression when sRNAs were up-regulated indicating that sRNAs are likely playing a negative regulatory role on mRNA targets at the transcript level. Target enrichment of sRNAs included mRNAs involved in transposons, chemotaxis signaling, peptidase activity, transcription factors, and secondary metabolite regulation. We exposed 5 biological replicates of H. volcanii strain H53 liquid cultures to the oxidative stress agent H2O2. Initially, cultures were grown in 80 mL of Hv-YPC under optimal conditions to an OD of 0.4 (mid exponential phase). To ensure homogeneity, each replicate was subsequently split into two 40 mL cultures, one used for the non-challenged (control) condition and the other for the oxidative stress condition (treatment). For the latter condition, 2 mM H2O2 (80% survival rate, previously determined) was directly added to the cultures followed by an hour incubation at 42 °C with shaking at 220 rpm. Total RNA was extracted using the Zymo Quick-RNA Miniprep kit and was split into two pools for total RNA-seq and a custom sRNA-seq protocol. 20 µg of total RNA was size selected from 50-500 nucleotides on a 7% denaturing urea polyacrylamide gel for sRNA-seq. Both the size-selected RNA and total RNA were used to make strand-specific libraries using SMART-seq Ultralow RNA input kit (Takara). Only three replicates (each condition) were sequenced for total RNA-seq thus resulting in a total of 16 libraries (10 sRNA-seq, 6 mRNA-seq).