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PMID: 24150936 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases.

Nucleic acids research ·Vol. 42 ·No. 2 ·2014-01-00 ·Pages 1341-53

Niewoehner O, Jinek M, Doudna JA

Abstract

In many bacteria and archaea, small RNAs derived from clustered regularly interspaced short palindromic repeats (CRISPRs) associate with CRISPR-associated (Cas) proteins to target foreign DNA for destruction. In Type I and III CRISPR/Cas systems, the Cas6 family of endoribonucleases generates functional CRISPR-derived RNAs by site-specific cleavage of repeat sequences in precursor transcripts. CRISPR repeats differ widely in both sequence and structure, with varying propensity to form hairpin folds immediately preceding the cleavage site. To investigate the evolution of distinct mechanisms for the recognition of diverse CRISPR repeats by Cas6 enzymes, we determined crystal structures of two Thermus thermophilus Cas6 enzymes both alone and bound to substrate and product RNAs. These structures show how the scaffold common to all Cas6 endonucleases has evolved two binding sites with distinct modes of RNA recognition: one specific for a hairpin fold and the other for a single-stranded 5'-terminal segment preceding the hairpin. These findings explain how divergent Cas6 enzymes have emerged to mediate highly selective pre-CRISPR-derived RNA processing across diverse CRISPR systems.

MeSH Terms
Bacterial Proteins/chemistry,metabolism CRISPR-Associated Proteins/chemistry,metabolism CRISPR-Cas Systems Catalytic Domain Clustered Regularly Interspaced Short Palindromic Repeats Endonucleases/chemistry,metabolism Models, Molecular Protein Binding RNA/chemistry,metabolism RNA Cleavage Thermus thermophilus/enzymology
Chemicals
Bacterial Proteins CRISPR-Associated Proteins RNA Endonucleases
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Niewoehner Ole
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA, Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA, Department of Chemistry, University of California, Berkeley, California 94720, USA and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Jinek Martin
Doudna Jennifer A
References (44)
44 references, click to expand
  1. CRISPR-based adaptive immune systems.
    Curr Opin Microbiol. 2011 Jun;14(3):321-7 PMID: 21531607
  2. Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for antiviral defense (CASCADE).
    J Biol Chem. 2011 Jun 17;286(24):21643-56 PMID: 21507944
  3. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea.
    Nat Rev Genet. 2010 Mar;11(3):181-90 PMID: 20125085
  4. Structural basis for CRISPR RNA-guided DNA recognition by Cascade.
    Nat Struct Mol Biol. 2011 May;18(5):529-36 PMID: 21460843
  5. XDS.
    Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):125-32 PMID: 20124692
  6. Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage.
    Structure. 2011 Feb 9;19(2):257-64 PMID: 21300293
  7. ARP/wARP and automatic interpretation of protein electron density maps.
    Methods Enzymol. 2003;374:229-44 PMID: 14696376
  8. RNA hairpin loop stability depends on closing base pair.
    Nucleic Acids Res. 1993 Aug 11;21(16):3845-9 PMID: 7690127
  9. Mechanism of substrate selection by a highly specific CRISPR endoribonuclease.
    RNA. 2012 Apr;18(4):661-72 PMID: 22345129
  10. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats.
    BMC Bioinformatics. 2007 May 23;8:172 PMID: 17521438
  11. Sequence- and structure-specific RNA processing by a CRISPR endonuclease.
    Science. 2010 Sep 10;329(5997):1355-8 PMID: 20829488
  12. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids.
    Nucleic Acids Res. 2007 Jul;35(Web Server issue):W375-83 PMID: 17452350
  13. Recognition and maturation of effector RNAs in a CRISPR interference pathway.
    Nat Struct Mol Biol. 2011 Jun;18(6):688-92 PMID: 21572444
  14. RNA-guided genetic silencing systems in bacteria and archaea.
    Nature. 2012 Feb 15;482(7385):331-8 PMID: 22337052
  15. An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3.
    Nat Struct Mol Biol. 2011 Jun;18(6):680-7 PMID: 21572442
  16. Crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 reveals a new protein family with an RNA recognition motif-like domain.
    Protein Sci. 2006 Jun;15(6):1494-9 PMID: 16672237
  17. Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes.
    Biol Chem. 2011 Apr;392(4):277-89 PMID: 21294681
  18. CRISPR interference: a structural perspective.
    Biochem J. 2013 Jul 15;453(2):155-66 PMID: 23805973
  19. ESPript: analysis of multiple sequence alignments in PostScript.
    Bioinformatics. 1999 Apr;15(4):305-8 PMID: 10320398
  20. A genome-wide view of the expression and processing patterns of Thermus thermophilus HB8 CRISPR RNAs.
    RNA. 2012 Apr;18(4):783-94 PMID: 22355165
  21. Automated structure solution with the PHENIX suite.
    Methods Mol Biol. 2008;426:419-35 PMID: 18542881
  22. Using prime-and-switch phasing to reduce model bias in molecular replacement.
    Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2144-9 PMID: 15572767
  23. RNA-protein analysis using a conditional CRISPR nuclease.
    Proc Natl Acad Sci U S A. 2013 Apr 2;110(14):5416-21 PMID: 23493562
  24. PDBe: Protein Data Bank in Europe.
    Nucleic Acids Res. 2012 Jan;40(Database issue):D445-52 PMID: 22110033
  25. Structures of the RNA-guided surveillance complex from a bacterial immune system.
    Nature. 2011 Sep 21;477(7365):486-489 PMID: 21938068
  26. Recognition and cleavage of a nonstructured CRISPR RNA by its processing endoribonuclease Cas6.
    Structure. 2013 Mar 5;21(3):385-93 PMID: 23454186
  27. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions.
    Proc Natl Acad Sci U S A. 2011 Jun 21;108(25):10092-7 PMID: 21536913
  28. Towards automated crystallographic structure refinement with phenix.refine.
    Acta Crystallogr D Biol Crystallogr. 2012 Apr;68(Pt 4):352-67 PMID: 22505256
  29. Dali: a network tool for protein structure comparison.
    Trends Biochem Sci. 1995 Nov;20(11):478-80 PMID: 8578593
  30. Coot: model-building tools for molecular graphics.
    Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32 PMID: 15572765
  31. Evolutionary conservation of sequence and secondary structures in CRISPR repeats.
    Genome Biol. 2007;8(4):R61 PMID: 17442114
  32. PHENIX: a comprehensive Python-based system for macromolecular structure solution.
    Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21 PMID: 20124702
  33. Small CRISPR RNAs guide antiviral defense in prokaryotes.
    Science. 2008 Aug 15;321(5891):960-4 PMID: 18703739
  34. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems.
    Biol Direct. 2011 Jul 14;6:38 PMID: 21756346
  35. A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis.
    Nucleic Acids Res. 2002 Jan 15;30(2):482-96 PMID: 11788711
  36. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes.
    PLoS Comput Biol. 2005 Nov;1(6):e60 PMID: 16292354
  37. Automated structure solution with autoSHARP.
    Methods Mol Biol. 2007;364:215-30 PMID: 17172768
  38. Transcription profile of Thermus thermophilus CRISPR systems after phage infection.
    J Mol Biol. 2010 Jan 15;395(2):270-81 PMID: 19891975
  39. The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA complex.
    Protein Sci. 2012 Mar;21(3):405-17 PMID: 22238224
  40. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes.
    Genes Dev. 2008 Dec 15;22(24):3489-96 PMID: 19141480
  41. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex.
    Cell. 2009 Nov 25;139(5):945-56 PMID: 19945378
  42. Phaser crystallographic software.
    J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674 PMID: 19461840
  43. Evolution and classification of the CRISPR-Cas systems.
    Nat Rev Microbiol. 2011 Jun;9(6):467-77 PMID: 21552286
  44. Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR RNA.
    EMBO J. 2012 Jun 13;31(12):2824-32 PMID: 22522703
Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gkt922
Published
2014-01-00
Epub
2013-00-22
Pages
1341-53
Language
English
Region
England
NLM ID
0411011
PMCID
PMC3902920
Subset
IM
Grants
Howard Hughes Medical Institute · United States
Databases
PDB
4C8Y, 4C8Z, 4C97, 4C98, 4C9D
External Links
PubMed source DOI: 10.1093/nar/gkt922 PMC Full Text View journal details
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