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Small regulatory RNAs (sRNAs) are universally distributed in all three domains of life, Archaea, Bacteria, and Eukaryotes. In bacteria, sRNAs typically function by binding near the translation start site of their target mRNAs and thereby inhibit or activate translation. In eukaryotes, miRNAs and siRNAs typically bind to the 3′-untranslated region (3′-UTR) of their target mRNAs and influence translation efficiency and/or mRNA stability. In archaea, sRNAs have been identified in all species investigated using bioinformatic approaches, RNomics, and RNA-Seq. Their size can vary significantly between less than 50 to more than 500 nucleotides. Differential expression of sRNA genes has been studied using northern blot analysis, microarrays, and RNA-Seq. In addition, biological functions have been unraveled by genetic approaches, i.e., by characterization of designed mutants. As in bacteria, it was revealed that archaeal sRNAs are involved in many biological processes, including metabolic regulation, adaptation to extreme conditions, stress responses, and even in regulation of morphology and cellular behavior. Recently, the first target mRNAs were identified in archaea, including one sRNA that binds to the 5′-region of two mRNAs in Methanosarcina mazeiGö1 and a few sRNAs that bind to 3′-UTRs in Sulfolobus solfataricus, three Pyrobaculumspecies, and Haloferax volcanii, indicating that archaeal sRNAs appear to be able to target both the 5′-UTR or the 3′-UTRs of their respective target mRNAs. In addition, archaea contain tRNA-derived fragments (tRFs), and one tRF has been identified as a major ribosome-binding sRNA in H. volcanii, which downregulates translation in response to stress. Besides regulatory sRNAs, archaea contain further classes of sRNAs, e.g., CRISPR RNAs (crRNAs) and snoRNAs.

To define the complete sRNA population of the halophilic archaeon Haloferax volcanii, we employed high throughput sequencing. cDNAs were generated from RNA ranging in size from 17 to 500 nucleotides isolated from cells grown at three different conditions to exponential and stationary phase, respectively. Altogether, 145 intergenic and 45 antisense sRNAs were identified. Comparison of the expression profile showed different numbers of reads at the six different conditions for the majority of sRNAs. A striking difference in the number of sRNA reads was observed between cells grown under standard vs. low salt conditions. Furthermore, the six highest numbers of reads were found for low salt conditions. In contrast, only slight differences between sRNA reads at different growth temperatures were detected. Attempts to delete four sRNA genes revealed that one sRNA gene is essential. The three viable sRNA gene deletion mutants possessed distinct phenotypes. According to microarray analyses, the removal of the sRNA gene resulted in a profound change of the transcriptome when compared with the wild type. High throughput sequencing also showed the presence of high concentrations of tRNA derived fragments in H. volcanii. These tRF molecules were shown to have different amounts of reads at the six conditions analyzed. Northern analysis was used to confirm the presence of the tRNA-derived fragments.