Bioinformatic prediction and experimental verification of sRNAs in the haloarchaeon Haloferax volcanii.
Recently a small-scale RNomics study led to the experimental identification of 21 intergenic and 18 antisense sRNA genes in the haloarchaeon Haloferax volcanii. To broaden the knowledge about sRNAs in haloarchaea, two bioinformatic approaches were used to predict sRNA genes in the genome of H. volcanii. More than 120 putative intergenic sRNA genes were identified by these comparative genomic approaches. The expression of 61 of the predicted genes was analyzed using DNA microarrays, and 37 were found to be expressed under at least one of three conditions tested. Using the results of Northern blot analyses and of a high throughput sequencing study the number of expressed genes was raised to 54 and the small size was verified for 26 predicted sRNAs. An analysis of the coding capacity revealed that the set of predicted sRNAs most likely does not encode proteins or peptides. In two cases it turned out that the predictions had not identified bona fide sRNAs but conserved regions in UTRs of large protein-encoding transcripts. Taken together, the combination of bioinformatic prediction and experimental verification has more than tripled the number of known haloarchaeal sRNAs, underscoring the importance of regulatory RNAs in the third domain of life, the archaea. Further analyses of the biological functions of selected sRNAs, including the construction of deletion mutants, are currently under way.