title: A genome-wide view of the expression and processing patterns of Thermus thermophilus HB8 CRISPR RNAs.
abstract: The CRISPR-Cas system represents an RNA-based adaptive immune response system in prokaryotes and archaea. CRISPRs (clustered regularly interspaced short palindromic repeats) consist of arrays of short repeat-sequences interspaced by nonrepetitive short spacers, some of which show sequence similarity to foreign phage genetic elements. Their cistronic transcripts are processed to produce the mature CRISPR RNAs (crRNAs), the elements that confer immunity by base-pairing with exogenous nucleic acids. We characterized the expression and processing patterns of Thermus thermophilus HB8 CRISPRs by using differential deep-sequencing, which differentiates between 5' monophosphate and 5' non-monophosphate-containing RNAs and/or between 3' hydroxyl and 3' non-hydroxyl-containing RNAs. The genome of T. thermophilus HB8 encodes 11 CRISPRs, classified into three distinct repeat-sequence types, all of which were constitutively expressed without deliberately infecting the bacteria with phage. Analysis of the differential deep sequencing data suggested that crRNAs are generated by endonucleolytic cleavage, leaving fragments with 5' hydroxyl and 3' phosphate or 2',3'-cyclic phosphate termini. The 5' ends of all crRNAs are generated by site-specific cleavage 8 nucleotides upstream of the spacer first position; however, the 3' ends are generated by two alternative, repeat-sequence-type-dependent mechanisms. These observations are consistent with the operation of multiple crRNA processing systems within a bacterial strain.
authors.affiliation: Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA.
authors.forename: Stefan
authors.initials: S
authors.lastname: Juranek
authors.forename: Tali
authors.initials: T
authors.lastname: Eban
authors.forename: Yael
authors.initials: Y
authors.lastname: Altuvia
authors.forename: Miguel
authors.initials: M
authors.lastname: Brown
authors.forename: Pavel
authors.initials: P
authors.lastname: Morozov
authors.forename: Thomas
authors.initials: T
authors.lastname: Tuschl
authors.forename: Hanah
authors.initials: H
authors.lastname: Margalit
mesh_terms: D001483:Base Sequence; D015152:Blotting, Northern; D017931:DNA Primers; D016680:Genome, Bacterial; D010766:Phosphorylation; D012329:RNA, Bacterial / Q000235:genetics*; D016963:Thermus thermophilus / Q000235:genetics*
chemical_list: D017931:DNA Primers; D012329:RNA, Bacterial