Programmable RNA shredding by the type III-A CRISPR-Cas system of Streptococcus thermophilus

Mol Cell. 2014 Nov 20;56(4):506-17. doi: 10.1016/j.molcel.2014.09.027. Epub 2014 Nov 6.

Abstract

Immunity against viruses and plasmids provided by CRISPR-Cas systems relies on a ribonucleoprotein effector complex that triggers the degradation of invasive nucleic acids (NA). Effector complexes of type I (Cascade) and II (Cas9-dual RNA) target foreign DNA. Intriguingly, the genetic evidence suggests that the type III-A Csm complex targets DNA, whereas biochemical data show that the type III-B Cmr complex cleaves RNA. Here we aimed to investigate NA specificity and mechanism of CRISPR interference for the Streptococcus thermophilus Csm (III-A) complex (StCsm). When expressed in Escherichia coli, two complexes of different stoichiometry copurified with 40 and 72 nt crRNA species, respectively. Both complexes targeted RNA and generated multiple cuts at 6 nt intervals. The Csm3 protein, present in multiple copies in both Csm complexes, acts as endoribonuclease. In the heterologous E. coli host, StCsm restricts MS2 RNA phage in a Csm3 nuclease-dependent manner. Thus, our results demonstrate that the type III-A StCsm complex guided by crRNA targets RNA and not DNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • CRISPR-Associated Proteins / chemistry
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Associated Proteins / metabolism*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Endoribonucleases / genetics
  • Endoribonucleases / metabolism
  • Molecular Sequence Data
  • Protein Binding
  • Protein Structure, Quaternary
  • RNA Cleavage*
  • Scattering, Small Angle
  • Streptococcus thermophilus / enzymology
  • Streptococcus thermophilus / genetics*
  • X-Ray Diffraction

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • Endoribonucleases

Associated data

  • GENBANK/KM222358