title: The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease.
abstract: Prokaryotes are frequently exposed to potentially harmful invasive nucleic acids from phages, plasmids, and transposons. One method of defense is the CRISPR-Cas adaptive immune system. Diverse CRISPR-Cas systems form distinct ribonucleoprotein effector complexes that target and cleave invasive nucleic acids to provide immunity. The Type III-B Cmr effector complex has been found to target the RNA and DNA of the invader in the various bacterial and archaeal organisms where it has been characterized. Interestingly, the gene encoding the Csx1 protein is frequently located in close proximity to the Cmr1-6 genes in many genomes, implicating a role for Csx1 in Cmr function. However, evidence suggests that Csx1 is not a stably associated component of the Cmr effector complex, but is necessary for DNA silencing by the Cmr system in Sulfolobus islandicus. To investigate the function of the Csx1 protein, we characterized the activity of recombinant Pyrococcus furiosus Csx1 against various nucleic acid substrates. We show that Csx1 is a metal-independent, endoribonuclease that acts selectively on single-stranded RNA and cleaves specifically after adenosines. The RNA cleavage activity of Csx1 is dependent upon a conserved HEPN motif located within the C-terminal domain of the protein. This motif is also key for activity in other known ribonucleases. Collectively, the findings indicate that invader silencing by Type III-B CRISPR-Cas systems relies both on RNA and DNA nuclease activities from the Cmr effector complex as well as on the affiliated, trans-acting Csx1 endoribonuclease.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
authors.forename: Nolan F
authors.initials: NF
authors.lastname: Sheppard
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
authors.forename: Claiborne V C
authors.initials: CV
authors.lastname: Glover
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Terns
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA Department of Genetics, University of Georgia, Athens, Georgia 30602, USA Department of Microbiology, University of Georgia, Athens, Georgia 30602, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Terns
mesh_terms: D000241:Adenosine / Q000378:metabolism; D020816:Amino Acid Motifs; D019843:Archaeal Proteins; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D064113:CRISPR-Cas Systems; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000235:genetics* / Q000276:immunology; D019642:RNA, Archaeal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D016944:Sulfolobus / Q000235:genetics / Q000276:immunology
chemical_list: D019843:Archaeal Proteins; D064130:CRISPR-Associated Proteins; D019642:RNA, Archaeal; D011994:Recombinant Proteins; D004722:Endoribonucleases; D000241:Adenosine
keywords: CARF; CRISPR; Cas; Csx1; HEPN; ribonuclease