title: Characterization of CRISPR RNA biogenesis and Cas6 cleavage-mediated inhibition of a provirus in the haloarchaeon Haloferax mediterranei.
abstract: The adaptive immune system comprising CRISPR (clustered regularly interspaced short palindromic repeats) arrays and cas (CRISPR-associated) genes has been discovered in a wide range of bacteria and archaea and has recently attracted comprehensive investigations. However, the subtype I-B CRISPR-Cas system in haloarchaea has been less characterized. Here, we investigated Cas6-mediated RNA processing in Haloferax mediterranei. The Cas6 cleavage site, as well as the CRISPR transcription start site, was experimentally determined, and processing of CRISPR transcripts was detected with a progressively increasing pattern from early log to stationary phase. With genetic approaches, we discovered that the lack of Cas1, Cas3, or Cas4 unexpectedly resulted in a decrease of CRISPR transcripts, while Cas5, Cas6, and Cas7 were found to be essential in stabilizing mature CRISPR RNA (crRNA). Intriguingly, we observed a CRISPR- and Cas3-independent inhibition of a defective provirus, in which the putative Cascade (CRISPR-associated complex for antiviral defense) proteins (Cas5, Cas6, Cas7, and Cas8b) were indispensably required. A sequence carried by a proviral transcript was found to be homologous to the CRISPR repeat RNA and vulnerable to Cas6-mediated cleavage, implying a distinct interference mechanism that may account for this unusual inhibition. These results provide fundamental information for the subtype I-B CRISPR-Cas system in halophilic archaea and suggest diversified mechanisms and multiple physiological functions for the CRISPR-Cas system.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Li
authors.forename: Hailong
authors.initials: H
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Han
authors.forename: Jingfang
authors.initials: J
authors.lastname: Liu
authors.forename: Rui
authors.initials: R
authors.lastname: Wang
authors.forename: Dahe
authors.initials: D
authors.lastname: Zhao
authors.forename: Jian
authors.initials: J
authors.lastname: Zhou
authors.forename: Hua
authors.initials: H
authors.lastname: Xiang
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain; D019641:DNA, Archaeal / Q000235:genetics; D019848:Gene Expression Regulation, Archaeal / Q000502:physiology*; D020745:Genome, Archaeal; D019623:Haloferax mediterranei / Q000378:metabolism / Q000821:virology*; D020411:Oligonucleotide Array Sequence Analysis; D011533:Proviruses / Q000502:physiology*; D019642:RNA, Archaeal / Q000235:genetics / Q000378:metabolism*; D024363:Transcription Initiation Site
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D019642:RNA, Archaeal