High throughput sequencing reveals a plethora of small RNAs including tRNA derived fragments in Haloferax volcanii

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.