Regulation of the RNA and DNA nuclease activities required for Pyrococcus furiosus Type III-B CRISPR-Cas immunity

Kawanda Foster, Sabine Grüschow, Scott Bailey, Malcolm F. White, Michael P. Terns

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)
4 Downloads (Pure)

Abstract

Type III CRISPR-Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3' protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity.

Original languageEnglish
Pages (from-to)4418-4434
Number of pages17
JournalNucleic Acids Research
Volume48
Issue number8
Early online date21 Mar 2020
DOIs
Publication statusPublished - 7 May 2020

Keywords

  • EVOLUTIONARY CLASSIFICATION
  • SILENCING COMPLEX
  • CRYSTAL-STRUCTURE
  • I-G
  • CLEAVAGE
  • SYSTEMS
  • PROTEIN
  • DEFENSE
  • ENDORIBONUCLEASE
  • TRANSCRIPTION

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