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RNA. 2015 Jun;21(6):1147-58. doi: 10.1261/rna.049130.114. Epub 2015 Apr 22.

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus.

Author information

1
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
2
Department of Poultry Science, University of Georgia, Athens, Georgia 30602, USA.
3
Department of Genetics and Genome Sciences, Institute for Systems Genomics, University of Connecticut Health Center, Farmington, Connecticut 06030-6403, USA.
4
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA Department of Genetics, University of Georgia, Athens, Georgia 30602, USA Department of Microbiology, University of Georgia, Athens, Georgia 30602, USA.

Abstract

CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, and III) each target destruction of foreign nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr (Type III-B) Cas proteins associated with one of two size classes of crRNAs and cleaves complementary target RNAs. Here, we have isolated and characterized two additional native Pfu crRNPs containing either Csa (Type I-A) or Cst (Type I-G) Cas proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by mass spectrometry and immunoblotting and the crRNAs by RNA sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from all seven Pfu CRISPR loci and contain identical 5' ends (8-nt repeat-derived 5' tag sequences) but heterogeneous 3' ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3' end processing pathways following primary cleavage of common pre-crRNAs. Like other previously characterized Type I CRISPR-Cas effector complexes, we predict that the newly identified Pfu Csa and Cst crRNPs each function to target invading DNA, adding an additional layer of protection beyond that afforded by the previously characterized RNA targeting Cmr complex.

KEYWORDS:

CRISPR; Cas; Cmr; Csa; Cst; Pyrococcus furiosus

PMID:
25904135
PMCID:
PMC4436667
DOI:
10.1261/rna.049130.114
[Indexed for MEDLINE]
Free PMC Article

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