title: Conservation and variability in the structure and function of the Cas5d endoribonuclease in the CRISPR-mediated microbial immune system.
abstract: Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form an RNA-mediated microbial immune system against invading foreign genetic elements. Cas5 proteins constitute one of the most prevalent Cas protein families in CRISPR-Cas systems and are predicted to have RNA recognition motif (RRM) domains. Cas5d is a subtype I-C-specific Cas5 protein that can be divided into two distinct subgroups, one of which has extra C-terminal residues while the other contains a longer insertion in the middle of its N-terminal RRM domain. Here, we report crystal structures of Cas5d from Streptococcus pyogenes and Xanthomonas oryzae, which respectively represent the two Cas5d subgroups. Despite a common domain architecture consisting of an N-terminal RRM domain and a C-terminal β-sheet domain, the structural differences between the two Cas5d proteins are highlighted by the presence of a unique extended helical region protruding from the N-terminal RRM domain of X. oryzae Cas5d. We also demonstrate that Cas5d proteins possess not only specific endoribonuclease activity for CRISPR RNAs but also nonspecific double-stranded DNA binding affinity. These findings suggest that Cas5d may play multiple roles in CRISPR-mediated immunity. Furthermore, the specific RNA processing was also observed between S. pyogenes Cas5d protein and X. oryzae CRISPR RNA and vice versa. This cross-species activity of Cas5d provides a special opportunity for elucidating conserved features of the CRISPR RNA processing event.
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea.
authors.forename: Yoon
authors.initials: Y
authors.lastname: Koo
authors.forename: Donghyun
authors.initials: D
authors.lastname: Ka
authors.forename: Eun-Jin
authors.initials: EJ
authors.lastname: Kim
authors.forename: Nayoung
authors.initials: N
authors.lastname: Suh
authors.forename: Euiyoung
authors.initials: E
authors.lastname: Bae
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005658:Fungi / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D064130:CRISPR-Associated Proteins; D012313:RNA; D004247:DNA; D004722:Endoribonucleases
keywords: BME; CRISPR; Cas; Clustered regularly interspaced short palindromic repeats; EMSA; RNA recognition motif; RRM; TEV; crRNA; double-stranded DNA; double-stranded DNA binding; dsDNA; electrophoretic mobility shift assay; non-coding RNA; pre-crRNA; precursor CRISPR RNA; ribonuclease; small CRISPR RNA; tobacco etch virus; β-mercaptoethanol