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ORIGINAL RESEARCH article

Front. Mol. Biosci. | doi: 10.3389/fmolb.2021.640440

A small RNA is linking CRISPR-Cas and zinc transport Provisionally accepted The final, formatted version of the article will be published soon. Notify me

 Anita Marchfelder1*,  Pascal Märkle1, Lisa-Katharina Maier1,  Sandra Maaß2, Claudia Hirschfeld2,  Jürgen Bartel2,  Dörte Becher2 and  Björn Voß3
  • 1University of Ulm, Germany
  • 2University of Greifswald, Germany
  • 3University of Stuttgart, Germany

The function and mode of action of small regulatory RNAs is currently still understudied in archaea. In the halophilic archaeon H. 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 H. volcanii for 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 type Haloferax cells 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 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.

Keywords: sRNA (small RNA), Archaea, CRISPR-Cas, Zinc transport, haloarchaea

Received: 11 Dec 2020; Accepted: 01 Mar 2021.

Copyright: © 2021 Marchfelder, Märkle, Maier, Maaß, Hirschfeld, Bartel, Becher and Voß. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Anita Marchfelder, University of Ulm, Ulm, Germany, anita.marchfelder@uni-ulm.de