Binding and cleavage of CRISPR RNA by Cas6

Abstract

The CRISPR-Cas system provides many prokaryotes with acquired resistance to viruses and other mobile genetic elements. The core components of this defense system are small, host-encoded prokaryotic silencing (psi)RNAs and Cas (CRISPR-associated) proteins. Invader-derived sequences within the psiRNAs guide Cas effector proteins to recognize and silence invader nucleic acids. Critical for CRISPR-Cas defense is processing of the psiRNAs from the primary transcripts of the host CRISPR (clustered regularly interspaced short palindromic repeat) locus. Cas6, a previously identified endoribonuclease present in a wide range of prokaryotes with the CRISPR-Cas system, binds and cleaves within the repeat sequences that separate the individual invader targeting elements in the CRISPR locus transcript. In the present study, we investigated several key aspects of the mechanism of function of Cas6 in psiRNA biogenesis. RNA footprinting reveals that Pyrococcus furiosus Cas6 binds to a 7-nt (nucleotide) sequence near the 5' end of the CRISPR RNA repeat sequence, 14 nt upstream of the Cas6 cleavage site. In addition, analysis of the cleavage activity of P. furiosus Cas6 proteins with mutations at conserved residues suggests that a triad comprised of Tyr31, His46, and Lys52 plays a critical role in catalysis, consistent with a possible general acid-base RNA cleavage mechanism for Cas6. Finally, we show that P. furiosus Cas6 remains stably associated with its cleavage products, suggesting additional roles for Cas6 in psiRNA biogenesis.

FIGURE 1.

psiRNA biogenesis. The primary CRISPR locus transcript contains multiple guide sequences (color segments, G) separated by a repeat sequence (black segments, R). Cas6 cleaves within the repeats of a CRISPR transcript (cleavage site labeled with dotted line), eventually producing individual RNAs comprised of an 8-nt repeat sequence (i.e., psi-tag), ∼37-nt guide sequence, and 22-nt repeat sequence (1× intermediate). These “1× intermediate RNAs” are the end-products of Cas6 cleavage. The 22-nt repeat sequence found at the 3′ end of the 1× Cas6 product is subsequently removed from the mature psiRNAs (that guide the Cmr complex to cleave target RNAs in P. furiosus) by an unknown activity. Cas6 cleavage proceeds through a series of intermediates including the 2× intermediate shown here.

FIGURE 2.

Lead-induced and RNase A cleavage protection of CRISPR repeat RNA by P. furiosus Cas6. (A) 3′-end-labeled P. furiosus CRISPR repeat RNA was incubated in the absence (RNA) or presence of increasing concentrations of Cas6 (indicated as micromolar, μM) and subjected to RNase A cleavage (left panel) or lead-induced cleavage (right panel). RNAs were separated on 15% denaturing (7 M urea) polyacrylamide gels. Size markers include 5′-end-labeled RNA markers (M) and alkaline hydrolysis ladders (OH). (Blue bars) Sites of strong protection. (B) Cleavage protection assays performed as in A with 5′-end-labeled CRISPR repeat RNA. A summary of cleavage protections is displayed to the right of each gel. (C) A summary of observed cleavage protection is shown. The Cas6 cleavage site is indicated by an asterisk (*).

FIGURE 3.

Cleavage activity of Cas6 mutants. (A) Uniformly ³²P-labeled CRISPR repeat RNA was incubated in the absence (−) or presence of increasing concentrations of wild-type or mutant Cas6 (10, 50, and 500 nM) followed by separation on a 15% denaturing (7 M urea) polyacrylamide gel. The 5′ and 3′ cleavage products are indicated to the right. (B) Purified wild-type (wt) and mutant Cas6 proteins were analyzed by SDS-PAGE and Coomassie blue staining. Molecular weight markers are indicated in kilodaltons.

FIGURE 4.

Substrate binding activity of Cas6 mutants. Uniformly ³²P-labeled CRISPR repeat RNA was incubated in the absence (−) or presence of increasing concentrations of wild-type or mutant Cas6 (1, 50, 200, and 500 nM). Formation of stable complexes was assessed by native gel mobility shift analysis. The positions of the free (RNA) and bound (RNP) substrate RNA are indicated.

FIGURE 5.

Immunopurified Cas6 cleaves CRISPR repeat RNA and associates with substrate and product RNAs. (A) Cleavage activity. Uniformly ³²P-labeled CRISPR repeat RNA was incubated in the absence (RNA) or presence of recombinant Cas6 (rCas6), whole cell extract (WCE), or samples from immunoprecipitation reactions using Cas6 antibodies; (Pre) preimmune; (Imm) immune; (S) supernatant; (P) pellet. The RNAs were separated on a 15% denaturing, 7 M urea-containing polyacrylamide gel, along with 5′-end-labeled RNA markers (M). (B) Northern blot analysis of Cas6 immunoprecipitation. RNAs extracted from WCE or from IPs using preimmune (Pre) or immune (Imm) Cas6 or Cmr2 antibodies were separated on 15% denaturing, 7 M urea-containing polyacrylamide gels, along with 5′-end-labeled RNA markers (M). A 5′-end-labeled DNA oligonucleotide antisense to psiRNA 6.01-specific sequences, or to the repeat sequence of P. furiosus CRISPRs 1, 5, and 6 were used as probes, as indicated. The positions of the 2× intermediate (2×), 1× intermediate (1×), and mature psiRNAs (arrows) are indicated.