A small RNA is linking CRISPR-Cas and zinc transport

Abstract

AbstractThe function and mode of action of small regulatory RNAs is currently still understudied in archaea. In the halophilic archaeonH. volcaniia plethora of sRNAs have been identified, however, in-depth functional analysis is missing for most of them. We selected a small RNA (s479) fromH. volcaniifor detailed characterization. The sRNA gene is encoded between a CRISPR RNA locus and the Cas protein gene cluster, the s479 deletion strain is viable and was characterized in detail. Transcriptome studies of wild typeHaloferaxcells and the deletion mutant revealed up-regulation of six genes in the deletion strain, showing that the sRNA has a clearly defined function. Three of the six up-regulated genes encode potential zinc transporter proteins (ZnuA1, ZnuB1, ZnuC1) suggesting involvement of s479 in regulation of zinc transport. Upregulation of these genes in the deletion strain was confirmed by northern blot and proteome analyses. Furthermore, electrophoretic mobility shift assays demonstrate a direct interaction of s479 with the targetznuC1 mRNA. Proteome comparison of wild type and deletion strains further expanded the regulon of s479 deeply rooting this sRNA within the metabolism ofH. volcaniiespecially the regulation of transporter abundance. Interestingly, s479 is not only encoded next to CRISPR-casgenes but the mature s479 contains a crRNA-like 5’ handle and experiments with Cas protein deletion strains indicate maturation by Cas6 and interaction with Cas proteins. Together this might suggest that the CRISPR-Cas system is involved in s479 function.