title: Programmable RNA shredding by the type III-A CRISPR-Cas system of Streptococcus thermophilus.
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.
authors.affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania.
authors.forename: Gintautas
authors.initials: G
authors.lastname: Tamulaitis
authors.affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania.
authors.forename: Migle
authors.initials: M
authors.lastname: Kazlauskiene
authors.affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania.
authors.forename: Elena
authors.initials: E
authors.lastname: Manakova
authors.affiliation: Department of Bioinformatics, Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania.
authors.forename: Česlovas
authors.initials: Č
authors.lastname: Venclovas
authors.affiliation: Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Mappin Street, Sheffield S1 3JD, UK.
authors.forename: Alison O
authors.initials: AO
authors.lastname: Nwokeoji
authors.affiliation: Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Mappin Street, Sheffield S1 3JD, UK.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Dickman
authors.affiliation: DuPont Nutrition and Health, BP10, Dangé-Saint-Romain 86220, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Horvath
authors.affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania. Electronic address: siksnys@ibt.lt.
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D059367:RNA Cleavage; D053838:Scattering, Small Angle; D048249:Streptococcus thermophilus / Q000201:enzymology / Q000235:genetics*; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D004722:Endoribonucleases