Investigation of the cyclic oligoadenylate signaling pathway of type III CRISPR systems

Christophe Rouillon, Januka S. Athukoralage, Shirley Graham, Sabine Grüschow, Malcolm F. White*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

13 Citations (Scopus)

Abstract

Type III CRISPR effector complexes utilize a bound CRISPR RNA (crRNA) to detect the presence of RNA from invading mobile genetic elements in the cell. This RNA binding results in the activation of two enzymatic domains of the Cas10 subunit—the HD nuclease domain, which degrades DNA, and PALM/cyclase domain. The latter synthesizes cyclic oligoadenylate (cOA) molecules by polymerizing ATP, and cOA acts as a second messenger in the cell, switching on the antiviral response by activating host ribonucleases and other proteins. In this chapter, we focus on the methods required to study the biochemistry of this recently discovered cOA signaling pathway. We cover protein expression and purification, synthesis of cOA and its linear analogues, kinetic analysis of cOA synthesis and cOA-stimulated ribonuclease activity, and small molecule detection and identification with thin-layer chromatography and mass spectrometry. The methods described are based on our recent studies of the type III CRISPR system in Sulfolobus solfataricus, but are widely applicable to other type III systems.

Original languageEnglish
Title of host publicationMethods in Enzymology
EditorsScott Bailey
PublisherAcademic Press/Elsevier
Pages191-218
Number of pages28
ISBN (Print)9780128167601
DOIs
Publication statusPublished - 1 Jan 2019

Publication series

NameMethods in Enzymology
Volume616
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • CARF
  • CRISPR type III
  • Csm6
  • Csx1
  • Cyclic oligoadenylate
  • HEPN
  • MazEF
  • Ribonuclease
  • Ring nuclease

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