A complex of Cas proteins 5, 6, and 7 is required for the biogenesis and stability of clustered regularly interspaced short palindromic repeats (crispr)-derived rnas (crrnas) in Haloferax volcanii

Abstract

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1-8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA.

Keywords: Archaea; CRISPR/Cas; Cas6; Haloferax volcanii; Microbiology; Molecular Biology; Molecular Genetics; Protein Complexes; Type I-B; crRNA.

FIGURE 1.

Location of H. volcanii cas genes and CRISPR loci. The cas gene cluster is located on the minichromosome pHV4 and encodes the Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, and Cas8b proteins, the latter being the signature protein for subtype I-B. The cluster is flanked by two CRISPR RNA loci (P1 and P2). CRISPR locus P1 contains 17 repeats and 16 spacers, and locus P2 contains 12 repeats and 11 spacers. The third CRISPR RNA locus is encoded on the main chromosome and contains 25 repeats and 24 spacers. The leader region (L) at the 5′-end of the CRISPR locus contains the promoter. The 5′-end of the cas6 mRNA was mapped with 5′-RACE and is indicated in the box above the cas gene cluster by an arrow. The overlap of the cas6 and cas8b genes is shown in the box below the cas gene cluster.

FIGURE 2.

Alignment of Cas6 proteins. Cas6 proteins from Haloferax, H. lacusprofundi, M. maripaludis, P. furiosus, E. coli, and P. aeruginosa are aligned to identify conserved amino acids. Amino acids from M. maripaludis, P. furiosus, E. coli, and P. aeruginosa that were shown to be essential for catalysis are indicated by red boxes. The amino acids mutated in this study are indicated by asterisks. Amino acids shown to be important for the stable crRNA population in this study are boxed in red (mutations result in reduced crRNA amounts) or green (mutations result in higher crRNA amounts).

FIGURE 3.

The haloarchaeal CRISPR-Cas systems are different from other systems. The haloarchaeal CRISPR-Cas systems are distinct from published systems where the Cas6 protein has been functionally characterized. The circular hierarchical tree represents the sequence and structure similarity of repeats from all publicly available genomes, taken from the CRISPRmap Web server (39). The locations of repeats associated with previously characterized Cas6 are highlighted with red lines: Clostridium thermocellum (16), P. furiosus (10, 31, 32), E. coli (20), T. thermophilus (25, 27, 29), P. aeruginosa (21, 26), Nanoarchaeum equitans (65), Synechocystis (66), M. mazei (13), S. epidermidis (30), and M. maripaludis (16). The pairwise alignment percentage identities in comparison with the Cas6 protein in H. volcanii are given in square brackets. For the CRISPRmap tree, brown branches represent CRISPRs from bacteria, the blue-green branches represent CRISPRs from archaea, the inner annotation circle represents different conserved structure motifs, the middle circle represents conserved sequence families, and the outer circle represents the six superclasses.

FIGURE 4.

The cas6 deletion strain does not generate crRNAs. To determine the biological function of the Cas6 protein, we deleted the reading frame of this gene in H. volcanii and isolated RNA from the deletion (Δcas6) and wild type strains, which was subsequently separated on 8% PAGE and transferred to a membrane. Hybridization with a probe against spacer 1 from locus P1 showed that no crRNAs were generated in the Δcas6 strain. Complementation of this strain with the cas6 gene on a plasmid resulted in the rescue of crRNA production.

FIGURE 5.

Effect of cas6 gene mutations on crRNA amounts. Mutations were introduced into the cas6 gene (Fig. 2), and the cas6 deletion strain was transformed with the mutant genes. A and B, RNA from all strains was isolated, separated on 8% PAGE, and subsequently transferred to Northern membranes that were then hybridized with a probe against spacer 1 from locus P1. Determination of the amount of crRNA in relation to the amount of RNA loaded (measured by the 5 S rRNA hybridization) showed that only transformation with three variants (His-41, Gly-256, and Gly-258) resulted in reduced amounts of crRNAs. Higher amounts of crRNA were determined in mutants Ser-115 and Ser-224. On the left, a DNA size marker in nucleotides is shown. The hybridization with the spacer 1 from CRISPR locus P1 is shown at the top, and hybridization with a probe against 5 S rRNA is shown at the bottom. marker, the DNA size marker; wt, RNA from wild type cells; Δcas6, RNA from the cas6 gene deletion strain; Δcas6 + cas6, RNA from the cas6 gene deletion strain complemented with the wild type cas6 gene from a plasmid; +Y15A, +Y19A, +H21A, +K22A, +R26A, +W28A, +H41A, +H45A, +F49A, +Y51A, +R66A, +S115A, +T116A, +L172A, +S224A, +W226A, +G248A, +G250A, +G256A, +F257A, and +G258A, RNA from the cas6 deletion strain complemented with the mutant cas6 gene from a plasmid. C, model of the H. volcanii Cas6 protein. Similarity searches in the Phyre database (54) show the closest structures related to the H. volcanii Cas6 protein to be the P. furiosus Cas6 structure (32). The H. volcanii protein was modeled according to the published P. furiosus structure, and the amino acid mutations that changed the amounts of crRNA are shown. His-41 is located where the catalytic site in the Pfu Cas6 protein was proposed; according to this model, the two glycines and serines might be located on the surface where the crRNA could be located.

FIGURE 6.

Cas6 alone is not sufficient for crRNA processing and maintenance. A, constructs used for complementation. To determine which cas genes are required for crRNA maturation, different cas gene constructs were generated for transformation of the H26ΔcasCluster28 strain, which has the complete cas gene cluster deleted. B, effect of different Cas proteins on crRNA amounts. RNA isolated from the H26ΔcasCluster28 strains, transformed with the different cas gene combinations, was transferred to membranes that were subsequently hybridized with a probe against spacer 1 from CRISPR locus P1. On the left, a DNA size marker (in nucleotides) is shown. The hybridization with spacer 1 from CRISPR locus P1 is shown at the top, and hybridization with a probe against 5 S rRNA is shown at the bottom. marker, DNA size marker; wt, RNA from wild type cells; Δcas, RNA from the cas gene deletion strain; Δcas + 68, RNA from the cas gene deletion strain complemented with the cas6 and cas8b genes from a plasmid; +67, +65, +87, +85, +75, +675, +685, +687, and +6875, RNA from the cas gene deletion strain complemented with the different cas genes from a plasmid as indicated.

FIGURE 7.

A and B, Cas5 and Cas6 co-purify with a FLAG-tagged Cas7 protein. A FLAG-tagged Cas7 protein was generated in Haloferax cells and purified together with all potential interaction partners using the FLAG tag. The purified fraction was investigated using a Western blot (A) and SDS-PAGE (B). Three proteins are visible on the Coomassie-stained SDS gel. The largest protein is, according to the Western blot (which was probed with the FLAG antibody), the FLAG-Cas7 fusion protein. According to mass spectrometry analyses, the two smaller proteins are Cas5 and Cas6, respectively. A, Western blot of the FLAG-purified fraction, probed with an anti-FLAG antibody from a plasmid; B, silver-stained SDS-PAGE of the FLAG-purified fraction. The protein size marker is given on the left in kDa, and the nature of the respective proteins is indicated on the right. C, the Cascade I-B complex contains crRNA. RNA was isolated from the FLAG-Cas7 purified protein fraction, separated by 8% PAGE, and transferred to a membrane. Hybridization with a probe against spacer 1 from CRISPR locus P1 detected two RNAs of ∼68 nucleotides and 51 nucleotides in length, which correspond to the Haloferax crRNAs (42). Shown on the left is a DNA size marker in nucleotides.