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

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

Sonali Majumdar et al. RNA. 2015 Jun.

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.

© 2015 Majumdar et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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Figures

FIGURE 1.
FIGURE 1.

(A) Organization of cas genes in Pyrococcus furiosus. Cas genes are mainly distributed in two gene clusters in Pfu. Genes encoding predicted adaptation Cas proteins (Cas1, Cas2, Cas4, gray), crRNA biogenesis protein (Cas6, gray) and immune-effector modules: Type I-A (Csa, green), Type I-G (Cst, yellow), Type III-B (Cmr, blue) are labeled. Genes encoding Cas3 (light yellow) and Cas3′ (helicase or hel.), Cas3″ (nuclease or nuc.) (light green) correspond to their subtype association. Inverted Y indicates polyclonal antibodies were raised against the specified Csa, Cst, and Cmr proteins. (B) Western blotting of Pfu extract with Csa, Cst, and Cmr antibodies. The indicated antibodies detect a major band (asterisk) of the expected molecular weight for each antigen. The predicted molecular weights for each protein are Csa2 (37.4 kDa), Cas3″ (22.9 kDa), Cst2 (39.7 kDa), Cas5t (23.8 kDa), and Cmr2 (100.8 kDa).

FIGURE 2.
FIGURE 2.

Cas protein composition of immunopurified Csa, Cst, and Cmr complexes. (A) Cas proteins identified by tandem mass spectrometric analysis of Csa (green), Cst (yellow), Cmr (blue) immunoprecipitated complexes are indicated by a check mark. Some Cas proteins predicted to be part of a complex but not identified by mass spectrometry are also listed. (B) Silver-stained SDS-polyacrylamide gels of Csa (Csa2), Cst (Cst2), and Cmr (Cmr2) immunoprecipitated pellets with preimmune (PI) or immune (I) antibodies as labeled. Cas proteins identified by tandem mass spectrometry are indicated based on expected molecular weight. The antigens are labeled in bold. Cas proteins identified by mass spectrometry that do not correlate to a visible band are marked with an asterisk. M is the protein standard in kilodaltons (kDa).

FIGURE 3.
FIGURE 3.

Western blot analysis of Cas proteins associated with Csa, Cst, and Cmr complexes. All five immunopurified samples were blotted and probed with each of the five immune antibodies (Csa2, Cas3″, Cst2, Cas5t, Cmr2) individually. The first lane of each blot is untreated Pfu S100 extract used as a reference. An asterisk indicates the expected position of antigens based on their molecular weights. Molecular weights are indicated in kilodaltons (kDa).

FIGURE 4.
FIGURE 4.

Northern blot analysis of crRNAs associated with Csa, Cst, and Cmr complexes. Northern blotting of RNAs extracted from Pfu S100 extract (T), preimmune pellets (PI), and immune pellets (I) of Csa (Csa2, Cas3″), Cst (Cst2, Cas5t), and Cmr (Cmr2). The blots were probed for the first crRNA of locus 7 (7.01). Asterisk indicates the prominent 45-nt crRNA band detected in all three complexes. Radiolabeled decade marker in nucleotides (M).

FIGURE 5.
FIGURE 5.

RNA-seq profiles of crRNAs associated with the Csa, Cst, and Cmr complexes and in total RNA. The number of reads (in thousands) of crRNAs-associated with each complex (extracted from immune pellets of Cas3″, Csa2, Cas5t, Cst2, Cmr2) compared with crRNAs in total RNA (extracted from untreated S100 extract) that map to CRISPR locus 5 are shown. A higher resolution image of three crRNAs is highlighted in the box on the right. Reads mapping to both the sense (red, transcribed from upstream leader) and antisense (blue) strand are indicated. The black boxes at the top correspond to CRISPR repeats interspersed by guide sequences shown as black lines.

FIGURE 6.
FIGURE 6.

Mapping ends of crRNAs in total RNA and crRNAs associated with Csa, Cst, Cmr complexes. 5′ and 3′ end analysis of all sequenced crRNAs in untreated S100 extract (total RNA) and crRNAs associated with Csa (Csa2, Cas3″), Cst (Cst2, Cas5t), and Cmr (Cmr2) complexes. The percentage of crRNAs starting and ending at a specific point is marked on the y-axis. The nucleotides are counted relative to their position from the guide-repeat junction. The nucleotides upstream of the guide-repeat junction are counted from −1 and those downstream are counted from +1. The peaks in the 5′ repeat region are shown in blue. The peaks downstream from the 5′ repeat-guide junction are shown in orange. The schematic on top indicates where the peaks map (repeats black and guides green).

FIGURE 7.
FIGURE 7.

Model for integration of crRNAs into three distinct crRNP effector complexes. The CRISPR locus (repeat: black rectangles, invader-derived guide sequences: multiple colors) is transcribed from a promoter in the leader (brown) to form the pre-crRNA. Cas6 cleaves once in each repeat element of the pre-cRNA transcript to generate 1X RNA intermediates that each contain the 5′ tag, entire guide sequence, and 22 nt of 3′ repeat. Further 3′ end trimming by unknown mechanisms occurs either before, during, or after association with specific Cas proteins. The Cmr complex (blue) associates with two forms of crRNAs lacking any 3′ repeat. Csa (green) and Cst (yellow) complexes harbor crRNAs that contain variable amounts of 3′ repeat and one crRNA species without any 3′ repeat. Each crRNA size form can assemble into the corresponding effector complexes (one crRNA/complex) and guide targeting. The Cmr complex cleaves complementary RNA targets. The Csa and Cst effector complexes are predicted to target DNA homologous to the guide sequences in the crRNAs.

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