title: RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus.
abstract: CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1-Csm5) with an uneven stoichiometry and a single crRNA of variable size (35-53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence.
authors.affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB Wageningen, the Netherlands. Electronic address: raymond.staals@otago.ac.nz.
authors.forename: Raymond H J
authors.initials: RH
authors.lastname: Staals
authors.affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB Wageningen, the Netherlands.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Howard Hughes Medical Institute and California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720-3200, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Taylor
authors.affiliation: Howard Hughes Medical Institute and California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720-3200, USA.
authors.forename: Jack E
authors.initials: JE
authors.lastname: Kornfeld
authors.affiliation: Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Kundan
authors.initials: K
authors.lastname: Sharma
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, 3584 CH Utrecht, the Netherlands.
authors.forename: Arjan
authors.initials: A
authors.lastname: Barendregt
authors.affiliation: Laboratory of Systems and Synthetic Biology, Wageningen University, 6703 HB Wageningen, the Netherlands.
authors.forename: Jasper J
authors.initials: JJ
authors.lastname: Koehorst
authors.affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB Wageningen, the Netherlands.
authors.forename: Marnix
authors.initials: M
authors.lastname: Vlot
authors.affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB Wageningen, the Netherlands.
authors.forename: Nirajan
authors.initials: N
authors.lastname: Neupane
authors.affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB Wageningen, the Netherlands.
authors.forename: Koen
authors.initials: K
authors.lastname: Varossieau
authors.affiliation: RIKEN SPring-8 Center, Hyogo 679-5148, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Sakamoto
authors.affiliation: Global Research Cluster, RIKEN, Saitama 351-0198, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Global Research Cluster, RIKEN, Saitama 351-0198, Japan.
authors.forename: Naoshi
authors.initials: N
authors.lastname: Dohmae
authors.affiliation: Structural Biology Laboratory, RIKEN, Kanagawa 230-0045, Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.affiliation: Laboratory of Systems and Synthetic Biology, Wageningen University, 6703 HB Wageningen, the Netherlands.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Schaap
authors.affiliation: Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; Bioanalytics Research Group, Department of Clinical Chemistry, University Medical Center, 37075 Göttingen, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, 3584 CH Utrecht, the Netherlands.
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.affiliation: Howard Hughes Medical Institute and California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720-3200, USA; Department of Molecular and Cell Biology, Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720-3200, USA; Life Sciences Division, Lawrence Berkeley National Lab, Berkeley, CA 94720-3200, USA.
authors.forename: Eva
authors.initials: E
authors.lastname: Nogales
authors.affiliation: Howard Hughes Medical Institute and California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720-3200, USA; Department of Molecular and Cell Biology, Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720-3200, USA; Physical Biosciences Division, Lawrence Berkeley National Lab, Berkeley, CA 94720-3200, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
authors.affiliation: RIKEN SPring-8 Center, Hyogo 679-5148, Japan; Structural Biology Laboratory, RIKEN, Kanagawa 230-0045, Japan.
authors.forename: Akeo
authors.initials: A
authors.lastname: Shinkai
authors.affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB Wageningen, the Netherlands. Electronic address: john.vanderoost@wur.nl.
authors.forename: John
authors.initials: J
authors.lastname: van der Oost
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D059367:RNA Cleavage; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D012329:RNA, Bacterial; D004722:Endoribonucleases