CRISPR-Cas III-A Csm6 CARF Domain Is a Ring Nuclease Triggering Stepwise cA4 Cleavage with ApA>p Formation Terminating RNase Activity

Ning Jia, Roger Jones, Guangli Yang, Ouathek Ouerfelli, Dinshaw J. Patel

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

Type III-A CRISPR-Cas surveillance complexes containing multi-subunit Csm effector, guide, and target RNAs exhibit multiple activities, including formation of cyclic-oligoadenylates (cAn) from ATP and subsequent cAn-mediated cleavage of single-strand RNA (ssRNA) by the trans-acting Csm6 RNase. Our structure-function studies have focused on Thermococcus onnurineus Csm6 to deduce mechanistic insights into how cA4 binding to the Csm6 CARF domain triggers the RNase activity of the Csm6 HEPN domain and what factors contribute to regulation of RNA cleavage activity. We demonstrate that the Csm6 CARF domain is a ring nuclease, whereby bound cA4 is stepwise cleaved initially to ApApApA>p and subsequently to ApA>p in its CARF domain-binding pocket, with such cleavage bursts using a timer mechanism to regulate the RNase activity of the Csm6 HEPN domain. In addition, we establish T. onnurineus Csm6 as an adenosine-specific RNase and identify a histidine in the cA4 CARF-binding pocket involved in autoinhibitory regulation of RNase activity.

Original languageEnglish (US)
Pages (from-to)944-956.e6
JournalMolecular cell
Volume75
Issue number5
DOIs
StatePublished - Sep 5 2019

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Keywords

  • A CARF histidine contributes to autoinhibition
  • Csm6 CARF is a ring nuclease
  • Csm6 is an adenosine-specific RNase
  • Sequential cleavage bursts regulate RNase activity
  • Type III-A CRISPR-Cas Csm

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