RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus

Raymond H J Staals; Yifan Zhu; David W Taylor; Jack E Kornfeld; Kundan Sharma; Arjan Barendregt; Jasper J Koehorst; Marnix Vlot; Nirajan Neupane; Koen Varossieau; Keiko Sakamoto; Takehiro Suzuki; Naoshi Dohmae; Shigeyuki Yokoyama; Peter J Schaap; Henning Urlaub; Albert J R Heck; Eva Nogales; Jennifer A Doudna; Akeo Shinkai; John van der Oost

doi:10.1016/j.molcel.2014.10.005

RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus

Mol Cell. 2014 Nov 20;56(4):518-30. doi: 10.1016/j.molcel.2014.10.005. Epub 2014 Nov 6.

Authors

Raymond H J Staals¹, Yifan Zhu², David W Taylor³, Jack E Kornfeld³, Kundan Sharma⁴, Arjan Barendregt⁵, Jasper J Koehorst⁶, Marnix Vlot², Nirajan Neupane², Koen Varossieau², Keiko Sakamoto⁷, Takehiro Suzuki⁸, Naoshi Dohmae⁸, Shigeyuki Yokoyama⁹, Peter J Schaap⁶, Henning Urlaub¹⁰, Albert J R Heck⁵, Eva Nogales¹¹, Jennifer A Doudna¹², Akeo Shinkai¹³, John van der Oost¹⁴

Affiliations

¹ Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB Wageningen, the Netherlands. Electronic address: raymond.staals@otago.ac.nz.
² Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB Wageningen, the Netherlands.
³ Howard Hughes Medical Institute and California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720-3200, USA.
⁴ Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
⁵ Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, 3584 CH Utrecht, the Netherlands.
⁶ Laboratory of Systems and Synthetic Biology, Wageningen University, 6703 HB Wageningen, the Netherlands.
⁷ RIKEN SPring-8 Center, Hyogo 679-5148, Japan.
⁸ Global Research Cluster, RIKEN, Saitama 351-0198, Japan.
⁹ Structural Biology Laboratory, RIKEN, Kanagawa 230-0045, Japan.
¹⁰ 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.
¹¹ 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.
¹² 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.
¹³ RIKEN SPring-8 Center, Hyogo 679-5148, Japan; Structural Biology Laboratory, RIKEN, Kanagawa 230-0045, Japan.
¹⁴ Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB Wageningen, the Netherlands. Electronic address: john.vanderoost@wur.nl.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Bacterial Proteins / chemistry
Bacterial Proteins / metabolism*
Bacterial Proteins / ultrastructure
Base Sequence
CRISPR-Associated Proteins / chemistry
CRISPR-Associated Proteins / metabolism*
CRISPR-Associated Proteins / ultrastructure
Clustered Regularly Interspaced Short Palindromic Repeats*
Endoribonucleases / chemistry
Endoribonucleases / metabolism
Endoribonucleases / ultrastructure
Microscopy, Electron
Models, Molecular
Molecular Sequence Data
Protein Binding
Protein Structure, Quaternary
RNA Cleavage*
RNA, Bacterial / genetics
RNA, Bacterial / metabolism
Thermus thermophilus / enzymology
Thermus thermophilus / genetics*

Substances

Bacterial Proteins
CRISPR-Associated Proteins
RNA, Bacterial
Endoribonucleases

Grants and funding

HHMI/Howard Hughes Medical Institute/United States