Data_Sheet_1_A Small RNA Is Linking CRISPR–Cas and Zinc Transport.pdf

The function and mode of action of small regulatory RNAs is currently still understudied in archaea. In the halophilic archaeon Haloferax volcanii, a plethora of sRNAs have been identified; however, in-depth functional analysis is missing for most of them. We selected a small RNA (s479) from Haloferax volcanii for detailed characterization. The sRNA gene is encoded between a CRISPR RNA locus and the Cas protein gene cluster, and the s479 deletion strain is viable and was characterized in detail. Transcriptome studies of wild-type Haloferax cells and the deletion mutant revealed upregulation of six genes in the deletion strain, showing that this sRNA has a clearly defined function. Three of the six upregulated genes encode potential zinc transporter proteins (ZnuA1, ZnuB1, and ZnuC1) suggesting the involvement of s479 in the 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 target znuC1 mRNA. Proteome comparison of wild-type and deletion strains further expanded the regulon of s479 deeply rooting this sRNA within the metabolism of H. volcanii especially the regulation of transporter abundance. Interestingly, s479 is not only encoded next to CRISPR–cas genes, 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.

目前，古菌中小调控RNA的功能与作用机制仍未得到充分研究。在嗜盐古菌沃氏嗜盐盒菌（Haloferax volcanii）中，虽已鉴定出大量小RNA（sRNAs），但绝大多数尚未开展深入的功能分析。本研究选取沃氏嗜盐盒菌中的小RNA（s479）开展详细的功能表征。该sRNA基因定位于CRISPR RNA（crRNA）位点与Cas蛋白基因簇之间，s479缺失菌株具有生存活力，且已完成详细的特征分析。对野生型沃氏嗜盐盒菌细胞与缺失突变株的转录组学分析显示，缺失株中共有6个基因呈现表达上调，表明该sRNA具有明确的调控功能。上述6个上调基因中的3个编码潜在的锌转运蛋白（ZnuA1、ZnuB1与ZnuC1），提示s479参与锌转运的调控过程。Northern印迹（Northern blot）与蛋白质组学分析证实了缺失株中这些基因的表达上调现象。此外，电泳迁移率变动分析（electrophoretic mobility shift assay, EMSA）证实s479可与靶标znuC1 mRNA直接结合。野生型菌株与缺失株的蛋白质组比较分析进一步拓展了s479的调控子范围，表明该sRNA深度参与沃氏嗜盐盒菌的代谢过程，尤其是转运蛋白丰度的调控。值得注意的是，s479不仅定位于CRISPR–cas基因旁侧，其成熟转录本还包含类似crRNA的5'端柄结构；通过Cas蛋白缺失菌株开展的实验表明，s479由Cas6蛋白介导成熟，并可与Cas蛋白发生相互作用。综上，上述结果提示CRISPR-Cas系统参与了s479的功能发挥。