title: Target RNA capture and cleavage by the Cmr type III-B CRISPR-Cas effector complex.
abstract: The effector complex of the Cmr/type III-B CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) system cleaves RNAs recognized by the CRISPR RNA (crRNA) of the complex and includes six protein subunits of unknown functions. Using reconstituted Pyrococcus furiosus Cmr complexes, we found that each of the six Cmr proteins plays a critical role in either crRNA interaction or target RNA capture. Cmr2, Cmr3, Cmr4, and Cmr5 are all required for formation of a crRNA-containing complex detected by native gel electrophoresis, and the conserved 5' repeat sequence tag and 5'-OH group of the crRNA are essential for the interaction. Interestingly, capture of the complementary target RNA additionally requires both Cmr1 and Cmr6. In detailed functional studies, we determined that P. furiosus Cmr complexes cleave target RNAs at 6-nucleotide (nt) intervals in the region of complementarity, beginning 5 nt downstream from the crRNA tag and continuing to within ∼14 nt of the 3' end of the crRNA. Our findings indicate that Cmr3 recognizes the signature crRNA tag sequence (and depends on protein-protein interactions with Cmr2, Cmr4, and Cmr5), each Cmr4 subunit mediates a target RNA cleavage, and Cmr1 and Cmr6 mediate an essential interaction between the 3' region of the crRNA and the target RNA.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA;
authors.forename: Caryn R
authors.initials: CR
authors.lastname: Hale
authors.affiliation: Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, USA;
authors.forename: Alexis
authors.initials: A
authors.lastname: Cocozaki
authors.affiliation: Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, USA; Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, USA;
authors.forename: Hong
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA; mterns@bmb.uga.edu.
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, Department of Microbiology, University of Georgia, Athens, Georgia 30602, USA mterns@bmb.uga.edu.
authors.forename: Michael P
authors.identifier: http://orcid.org/0000-0001-8330-1789
authors.initials: MP
authors.lastname: Terns
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism; D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D059367:RNA Cleavage
chemical_list: D019843:Archaeal Proteins; D064130:CRISPR-Associated Proteins; D026901:Membrane Transport Proteins; D021122:Protein Subunits; D012313:RNA
keywords: CRISPR; Cas; Cmr; Pyrococcus furiosus; RNAi; crRNA