To elucidate the role of small noncoding RNAs (sRNAs) in archaea we applied RNomics to identify sRNAs in the halophilic archaeon Haloferax volcanii. Using a size-selected cDNA library, 39 different previously uncharacterized sRNAs were identified ranging in size from 130 to 460 nucleotides. Twenty-one of these sRNAs are located in intergenic regions and 18 in antisense orientation. One of the intergenic sRNAs codes for a peptide. Only a minor fraction of sRNA genes were preceded by promoter elements (15 of 39), indicating that the majority might be generated by processing from larger precursors. Northern blot analyses of the intergenic sRNAs revealed differential expression for several sRNAs. Deletion mutants of two sRNAs were constructed, demonstrating that this approach is suitable to elucidate their biological function. Both mutant strains showed a defined phenotype: sRNA(30) gene deletion mutant was less resistant to higher temperatures and sRNA(63) gene deletion mutant resulted in a severe growth defect at low salt concentrations. Proteome analyses revealed clear differences between wildtype and deletion strains. These results represent the first reported examples of experimentally characterizing the function of sRNAs, excepting snoRNAs, in archaea. Taken together, we showed that haloarchaea encode sRNAs, some of which are differentially expressed and which have the potential to fulfil important biological functions in vivo.
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