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

Abstract

Haloarchaea in their natural environment are exposed to hypersalinity, intense solar radiation, and desiccation, all of which generate high levels of oxidative stress. Previous work has shown that haloarchaea are an order of magnitude more resistant to oxidative stress than most mesophilic organisms. Despite this resistance, the pathways haloarchaea use to respond to oxidative stress damage are similar to those of nonresistant organisms, suggesting that regulatory processes might be key to their robustness. Recently, small regulatory noncoding RNAs (sRNAs) were discovered in Archaea under a variety of environmental conditions. We report here the transcriptional landscape and functional roles of sRNAs in the regulation of the oxidative stress response of the model haloarchaeon Haloferax volcanii. Thousands of sRNAs, both intergenic and antisense, were discovered using strand-specific sRNA sequencing (sRNA-seq), comprising 25 to 30% of the total transcriptome under no-challenge and oxidative stress conditions, respectively. We identified hundreds of differentially expressed sRNAs in response to hydrogen peroxide-induced oxidative stress in H. volcanii. The targets of a group of antisense sRNAs decreased in expression when these sRNAs were upregulated, suggesting that sRNAs are potentially playing a negative regulatory role on mRNA targets at the transcript level. Target enrichment of these antisense sRNAs included mRNAs involved in transposon mobility, chemotaxis signaling, peptidase activity, and transcription factors.IMPORTANCE While a substantial body of experimental work has been done to uncover the functions of small regulatory noncoding RNAs (sRNAs) in gene regulation in Bacteria and Eukarya, the functional roles of sRNAs in Archaea are still poorly understood. This study is the first to establish the regulatory effects of sRNAs on mRNAs during the oxidative stress response in the haloarchaeon Haloferax volcanii. Our work demonstrates that common principles for the response to a major cellular stress exist across the 3 domains of life while uncovering pathways that might be specific to the Archaea. This work also underscores the relevance of sRNAs in adaptation to extreme environmental conditions.

Regulatory Noncoding Small RNAs Are Diverse and Abundant in an Extremophilic Microbial Community

“…In the halite community, we found that asRNA expression levels were negatively correlated with those of their putative targets, with highly expressed asRNAs overlapping lowly expressed protein-encoding genes. A similar trend was reported in the haloarchaeon H. volcanii when investigating oxidative stress-responsive sRNAs, and most of the putative targets were transposase genes (21). Putative target gene func-tions in our study were mostly from haloarchaea and enriched for transport systems, cell membrane, and cell wall metabolism, with a large number of hypotheticals.…”

“…SnapT for sRNA community identification. An analytic pipeline, SnapT for Small ncRNA Annotation Pipeline for (meta)Transcriptomic data, was adapted from our previous work (21) to find, annotate, and quantify intergenic and antisense sRNA transcripts from transcriptomic or metatranscriptomic data. In brief, de novo transcripts were assembled from RNA reads mapped to the metagenomic assembly, and transcripts that could not be explained by any protein-coding region and did not encode peptides were extracted and further validated as sRNAs.…”

“…The main source of liquid water for this community is from salt deliquescence (19), and it is sustained by CO 2 fixed via photosynthesis (16,20). While previous studies have demonstrated the role of sRNAs in the stress response of one of the members of this community, the halophilic archaeon Haloferax volcanii (21,22), there is no information on any of the other members.…”

“…Here, we used a combination of genome-resolved metagenomics and metatranscriptomics to investigate the role of sRNAs in the adaptive response of microorganisms inhabiting halite nodules. We developed an analytical pipeline, SnapT, built on our previous work on sRNAs from the archaeon H. volcanii (21), to enable the discovery of sRNAs at the community level. Using strand-specific metatranscriptomics, we found hundreds of sRNAs (both itsRNAs and asRNAs) from multiple trophic levels in the halite community, including conserved sRNAs, validating our experimental approach.…”

Regulatory non-coding small RNAs are diverse and abundant in an extremophilic microbial community

“…We used a read count-based differential expression analysis to identify differentially 133 expressed sRNA and mRNA transcripts. The program featureCounts (Liao,Smyth,and 134 Shi 2014) was used to rapidly count reads that map to the assembled RNA transcripts 135 (described above) as previously described (Gelsinger and DiRuggiero 2018a). In order 136 to account for organism abundance changes (as opposed to true transcript changes), 137…”