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. 2019 Apr;16(4):492-503.
doi: 10.1080/15476286.2018.1514234. Epub 2018 Sep 13.

Cross-cleavage Activity of Cas6b in crRNA Processing of Two Different CRISPR-Cas Systems in Methanosarcina Mazei Gö1

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Free PMC article

Cross-cleavage Activity of Cas6b in crRNA Processing of Two Different CRISPR-Cas Systems in Methanosarcina Mazei Gö1

Lisa Nickel et al. RNA Biol. .
Free PMC article

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR) system is a prokaryotic adaptive defense system against foreign nucleic acids. In the methanoarchaeon Methanosarcina mazei Gö1, two types of CRISPR-Cas systems are present (type I-B and type III-C). Both loci encode a Cas6 endonuclease, Cas6b-IB and Cas6b-IIIC, typically responsible for maturation of functional short CRISPR RNAs (crRNAs). To evaluate potential cross cleavage activity, we biochemically characterized both Cas6b proteins regarding their crRNA binding behavior and their ability to process pre-crRNA from the respective CRISPR array in vivo. Maturation of crRNA was studied in the respective single deletion mutants by northern blot and RNA-Seq analysis demonstrating that in vivo primarily Cas6b-IB is responsible for crRNA processing of both CRISPR arrays. Tentative protein level evidence for the translation of both Cas6b proteins under standard growth conditions was detected, arguing for different activities or a potential non-redundant role of Cas6b-IIIC within the cell. Conservation of both Cas6 endonucleases was observed in several other M. mazei isolates, though a wide variety was displayed. In general, repeat and leader sequence conservation revealed a close correlation in the M. mazei strains. The repeat sequences from both CRISPR arrays from M. mazei Gö1 contain the same sequence motif with differences only in two nucleotides. These data stand in contrast to all other analyzed M. mazei isolates, which have at least one additional CRISPR array with repeats belonging to another sequence motif. This conforms to the finding that Cas6b-IB is the crucial and functional endonuclease in M. mazei Gö1. Abbreviations: sRNA: small RNA; crRNA: CRISPR RNA; pre-crRNAs: Precursor CRISPR RNA; CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR associated; nt: nucleotide; RNP: ribonucleoprotein; RBS: ribosome binding site.

Keywords: CRISPR-Cas system; Cas6 endonucleases; Methanoarchaea; immunity of prokaryotes; regulatory RNA.

Figures

Figure 1.
Figure 1.
Size-exclusion chromatography of the Cas6b endonucleases of M. mazei Gö1 in the absence and presence of repeat RNA substrate. Size-exclusion chromatography on a Superose12 column (GE Healthcare) was performed with MBP-fusion protein Cas6b-IB (a) and Cas6b-IIIC (b) from M. mazei Gö1 (17 µM). Absorbance at 280 nm is shown for the respective proteins in blue and in red for a sample containing the respective protein pre-incubated in the presence of the deoxy-repeat-IB variant (5 µM). Gel filtration was performed with a linear flow rate of 0.5 ml/min using buffer A (see Material and Methods) and elution volume (ml) is indicated. Potential co-eluting RNA was identified via northern dot blot analysis using a 5ʹ radioactive labeled repeat-IB probe. Calibration of the column was performed using the gel-filtration mass standard (Bio-Rad Laboratories) containing thyroglobulin (670 kDa), IgG (158 kDa), ovalbumin (44 kDa), myoglobulin (17 kDa) and vitamin B12 (1.35 kDa).
Figure 2.
Figure 2.
crRNA abundance in M. mazei cas6b deletion strains. Total RNA was isolated from the M. mazei wild type strain and the deletion strains ∆cas6b-IB and ∆cas6b-IIIC growing under standard and high salt conditions (500 mM NaCl) as described in the Materials and Methods. Subsequent northern blot analysis was performed, using a 5ʹ radioactively labeled subtype IB-repeat probe. In addition, the expression of 5S rRNA of the respective RNA preparation is shown. The pUC mix8 standard marker (Thermo Scientific) was used (fragment sizes are indicated at the left side). On the right side, observed crRNA processing is depicted schematically.
Figure 3.
Figure 3.
crRNA processing in M. mazei wt and cas6b deletion strains for CRISPR-Cas subtypes I-B and III-C analyzed by an RNA-Seq approach. Normalized sequencing reads (HiSeq) for the CRISPR array subtype I-B (a) and subtype III-C (b) regions are mapped to the M. mazei genome. Comparison of analyzed RNA-Seq reads derived from exponentially growing cultures from wt and respective M. mazei deletion strains ∆cas6b-IB and ∆cas6b-IIIC. Cells were grown under high salt conditions (500 mM NaCl) and total RNA was treated with T4 polynucleotide kinase (PNK) as described in Materials and Methods.
Figure 4.
Figure 4.
crRNA 5ʹ and 3ʹ end processing on single nucleotide level in the M. mazei cas6b deletion strains. Normalized RNA-Seq reads for the CRISPR array subtype I-B and subtype III-C are mapped to the M. mazei genome. Comparison of analyzed RNA-Seq reads derived from the wt and from the M. mazei deletion strains ∆cas6b-IB and ∆cas6b-IIIC. Cells were grown under high salt conditions (500 mM NaCl) as described in Materials and Methods. Depicted is the crRNA containing spacer 4 from the subtype-IB array and the crRNA for the spacer 8 belonging to the subtype-IIIC array. The crRNA processing is shown on single nucleotide level, with one bar representing a single nucleotide. The position of spacer (grey box) and repeat (dark grey line) sequences are indicated according to the RNA-Seq reads.
Figure 5.
Figure 5.
Repeat and leader conservation among different M. mazei strains. The repeat (a) and leader (b) sequences from different free accessible genomes of M. mazei strains (Gö1, SarPi, WWM610, LYC, C16, Tuc01 and S-6) were extracted and a phylogenetic tree was generated based on sequence conservation. Conserved repeat motifs (Weblogo [49],) of each cluster are shown.
Figure 6.
Figure 6.
Cas6 conservation among different M. mazei strains. The Cas6 protein sequences from different free accessible genomes of M. mazei strains (Gö1, SarPi, WWM610, LYC, C16, Tuc01 and S-6) were extracted and (A) a heatmap and (B) a hierarchical cluster tree based on amino acid sequence conservation was generated. The both Cas6 proteins from M. mazei Gö1 are highlighted in red.

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This work was supported by the Deutsche Forschungsgemeinschaft [BA-2186/5-2 and Schm1052/12-2].

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