title: Characterization of CRISPR RNA processing in Clostridium thermocellum and Methanococcus maripaludis.
abstract: The CRISPR arrays found in many bacteria and most archaea are transcribed into a long precursor RNA that is processed into small clustered regularly interspaced short palindromic repeats (CRISPR) RNAs (crRNAs). These RNA molecules can contain fragments of viral genomes and mediate, together with a set of CRISPR-associated (Cas) proteins, the prokaryotic immunity against viral attacks. CRISPR/Cas systems are diverse and the Cas6 enzymes that process crRNAs vary between different subtypes. We analysed CRISPR/Cas subtype I-B and present the identification of novel Cas6 enzymes from the bacterial and archaeal model organisms Clostridium thermocellum and Methanococcus maripaludis C5. Methanococcus maripaludis Cas6b in vitro activity and specificity was determined. Two complementary catalytic histidine residues were identified. RNA-Seq analyses revealed in vivo crRNA processing sites, crRNA abundance and orientation of CRISPR transcription within these two organisms. Individual spacer sequences were identified with strong effects on transcription and processing patterns of a CRISPR cluster. These effects will need to be considered for the application of CRISPR clusters that are designed to produce synthetic crRNAs.
authors.affiliation: Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch Strasse 10, D-35037 Marburg, Germany.
authors.forename: Hagen
authors.initials: H
authors.lastname: Richter
authors.forename: Judith
authors.initials: J
authors.lastname: Zoephel
authors.forename: Jeanette
authors.initials: J
authors.lastname: Schermuly
authors.forename: Daniel
authors.initials: D
authors.lastname: Maticzka
authors.forename: Rolf
authors.initials: R
authors.lastname: Backofen
authors.forename: Lennart
authors.initials: L
authors.lastname: Randau
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D055029:Inverted Repeat Sequences; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012323:RNA Processing, Post-Transcriptional; D019642:RNA, Archaeal / Q000737:chemistry / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D019642:RNA, Archaeal; D012329:RNA, Bacterial; D006639:Histidine; D004722:Endoribonucleases