RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions

Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-associated (cas) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. Here we show that the Csy proteins (Csy1–4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Target recognition is enthalpically driven and localized to a “seed sequence” at the 5′ end of the CRISPR RNA spacer. Structural analysis of the complex by small-angle X-ray scattering and single particle electron microscopy reveals a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-associated complex from Escherichia coli, termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems.

[1]  Li Li,et al.  Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial , 2006, Genome Biology.

[2]  N. Grishin,et al.  A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action , 2006, Biology Direct.

[3]  Dmitri I. Svergun,et al.  Electronic Reprint Applied Crystallography Dammif, a Program for Rapid Ab-initio Shape Determination in Small-angle Scattering Applied Crystallography Dammif, a Program for Rapid Ab-initio Shape Determination in Small-angle Scattering , 2022 .

[4]  L. Marraffini,et al.  CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA , 2008, Science.

[5]  Dmitri I. Svergun,et al.  Determination of the regularization parameter in indirect-transform methods using perceptual criteria , 1992 .

[6]  Patrice Gouet,et al.  ESPript: analysis of multiple sequence alignments in PostScript , 1999, Bioinform..

[7]  I. Tanaka,et al.  Crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 reveals a new protein family with an RNA recognition motif‐like domain , 2006, Protein science : a publication of the Protein Society.

[8]  D. Barford,et al.  Enhancement of the Seed-Target Recognition Step in RNA Silencing by a PIWI/MID Domain Protein , 2009, Molecular cell.

[9]  B. Graveley,et al.  RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex , 2009, Cell.

[10]  J. García-Martínez,et al.  Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements , 2005, Journal of Molecular Evolution.

[11]  Dmitri I. Svergun,et al.  Uniqueness of ab initio shape determination in small-angle scattering , 2003 .

[12]  V. Kunin,et al.  Evolutionary conservation of sequence and secondary structures in CRISPR repeats , 2007, Genome Biology.

[13]  Daniel H. Haft,et al.  A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes , 2005, PLoS Comput. Biol..

[14]  Wilko Keegstra,et al.  Automation of specimen selection and data acquisition for protein electron crystallography , 1998 .

[15]  Jennifer A. Doudna,et al.  Sequence- and Structure-Specific RNA Processing by a CRISPR Endonuclease , 2010, Science.

[16]  Albert J R Heck,et al.  Structural basis for CRISPR RNA-guided DNA recognition by Cascade , 2011, Nature Structural &Molecular Biology.

[17]  Erik J. Sontheimer,et al.  Self vs. non-self discrimination during CRISPR RNA-directed immunity , 2009, Nature.

[18]  A. Heck,et al.  The effect of the source pressure on the abundance of ions of noncovalent protein assemblies in an electrospray ionization orthogonal time-of-flight instrument. , 2001, Rapid communications in mass spectrometry : RCM.

[19]  Philippe Horvath,et al.  The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA , 2010, Nature.

[20]  J. García-Martínez,et al.  Short motif sequences determine the targets of the prokaryotic CRISPR defence system. , 2009, Microbiology.

[21]  Stan J. J. Brouns,et al.  Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes , 2008, Science.

[22]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[23]  J. Mccammon,et al.  Situs: A package for docking crystal structures into low-resolution maps from electron microscopy. , 1999, Journal of structural biology.

[24]  R. Terns,et al.  Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. , 2008, RNA.

[25]  Stan J. J. Brouns,et al.  CRISPR-based adaptive and heritable immunity in prokaryotes. , 2009, Trends in biochemical sciences.

[26]  C. V. Jongeneel,et al.  The M-Coffee web server: a meta-method for computing multiple sequence alignments by combining alternative alignment methods , 2007, Nucleic Acids Res..

[27]  R. Terns,et al.  Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. , 2008, Genes & development.

[28]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.

[29]  Philippe Horvath,et al.  Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus , 2007, Journal of bacteriology.

[30]  Albert J R Heck,et al.  Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry. , 2006, Analytical chemistry.

[31]  M. Heel,et al.  Single-particle electron cryo-microscopy: towards atomic resolution , 2000, Quarterly Reviews of Biophysics.

[32]  Anders F. Andersson,et al.  Virus Population Dynamics and Acquired Virus Resistance in Natural Microbial Communities , 2008, Science.

[33]  K. Zhou,et al.  Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense. , 2009, Structure.

[34]  R. Barrangou,et al.  CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes , 2007, Science.

[35]  S. Ehrlich,et al.  Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. , 2005, Microbiology.