Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6.

Ole Niewoehner,Martin Jinek

doi:10.1261/rna.054098.115

Abstract

Prokaryotic CRISPR–Cas systems provide an RNA-guided mechanism for genome defense against mobile genetic elements such as viruses and plasmids. In type III-A CRISPR–Cas systems, the RNA-guided multisubunit Csm effector complex targets both single-stranded RNAs and double-stranded DNAs. In addition to the Csm complex, efficient anti-plasmid immunity mediated by type III-A systems also requires the CRISPR-associated protein Csm6. Here we report the crystal structure of Csm6 from Thermus thermophilus and show that the protein is a ssRNA-specific endoribonuclease. The structure reveals a dimeric architecture generated by interactions involving the N-terminal CARF and C-terminal HEPN domains. HEPN domain dimerization leads to the formation of a composite ribonuclease active site. Consistently, mutations of invariant active site residues impair catalytic activity in vitro. We further show that the ribonuclease activity of Csm6 is conserved across orthologs, suggesting that it plays an important functional role in CRISPR–Cas systems. The dimer interface of the CARF domains features a conserved electropositive pocket that may function as a ligand-binding site for allosteric control of ribonuclease activity. Altogether, our work suggests that Csm6 proteins provide an auxiliary RNA-targeting interference mechanism in type III-A CRISPR–Cas systems that operates in conjunction with the RNA- and DNA-targeting endonuclease activities of the Csm effector complex.

Highlights

IntroductionIn many bacteria and most archaea, CRISPR–Cas systems (for clustered regularly interspaced short palindromic repeats—CRISPR-associated) provide adaptive and heritable immunity against mobile genetic elements such as viruses and plasmids (Barrangou et al 2007; Marraffini and Sontheimer 2008, 2010; Wiedenheft et al 2012; Sorek et al 2013; van der Oost et al 2014)
In many bacteria and most archaea, CRISPR–Cas systems provide adaptive and heritable immunity against mobile genetic elements such as viruses and plasmids (Barrangou et al 2007; Marraffini and Sontheimer 2008, 2010; Wiedenheft et al 2012; Sorek et al 2013; van der Oost et al 2014)
The HEPN domains in the T. thermophilus Csm6 (TtCsm6) dimer form a composite ribonuclease active site whose architecture and function is likely conserved in other Csm6 proteins. These results suggest that besides the intrinsic RNA cleavage activity of the Csm effector complex mediated by its Csm3 subunits, type III-A CRISPR–Cas systems harbor an additional ribonuclease module—Csm6—whose activity may play an important role in the interference mechanism

Summary

Introduction

In many bacteria and most archaea, CRISPR–Cas systems (for clustered regularly interspaced short palindromic repeats—CRISPR-associated) provide adaptive and heritable immunity against mobile genetic elements such as viruses and plasmids (Barrangou et al 2007; Marraffini and Sontheimer 2008, 2010; Wiedenheft et al 2012; Sorek et al 2013; van der Oost et al 2014) These RNA-guided genome defense systems typically consist of an array of short repeats intercalated with invader-derived spacer sequences, and an operon containing several CRISPR-associated (cas) genes encoding the molecular machinery involved in spacer acquisition, guide RNA processing, and target interference. Type III CRISPR–Cas systems are defined by the signature protein Cas and, in analogy with type I systems, rely on multisubunit crRNA–Cas protein

Methods

Results

Conclusion