Sequence- and structure-specific RNA processing by a CRISPR endonuclease

Abstract

Many bacteria and archaea contain clustered regularly interspaced short palindromic repeats (CRISPRs) that confer resistance to invasive genetic elements. Central to this immune system is the production of CRISPR-derived RNAs (crRNAs) after transcription of the CRISPR locus. Here, we identify the endoribonuclease (Csy4) responsible for CRISPR transcript (pre-crRNA) processing in Pseudomonas aeruginosa. A 1.8 angstrom crystal structure of Csy4 bound to its cognate RNA reveals that Csy4 makes sequence-specific interactions in the major groove of the crRNA repeat stem-loop. Together with electrostatic contacts to the phosphate backbone, these enable Csy4 to bind selectively and cleave pre-crRNAs using phylogenetically conserved serine and histidine residues in the active site. The RNA recognition mechanism identified here explains sequence- and structure-specific processing by a large family of CRISPR-specific endoribonucleases.

Fig. 1. Csy4 specifically cleaves only its cognate pre-crRNA substrate

(A) Schematic of the CRISPR/Cas locus in Pa14. Two CRISPR loci flank the six Cas genes. Enlarged is a schematic showing the predicted stem-loop in the 28-nucleotide direct repeat (black lettering) separated by a 32-nucleotide unique spacer sequence (blue). Red arrows denote the cleavage site. (B) Csy4 (lanes 2–6, 8) was incubated with in vitro transcribed Pa14 pre-crRNA (lanes 1–6) for 30 seconds (lane 2), one minute (lane 3) and five minutes (lanes 4–6) in buffer containing no exogenous metal ions (lanes 2–4) and in buffer supplemented with 2.5 mM MgCl₂ (lane 5) or 2.5 mM EDTA (lane 6). Csy4 was also incubated with in vitro transcribed pre-crRNA from Streptococcus thermophilus (lanes 7–8) for 5 minutes (lane 8). Products were acid phenolchloroform extracted, separated on 15% denaturing PAGE and visualized with SYBR Gold staining. (C) Csy4 was heterologously expressed in E. coli in the presence (+) or absence (−) of a plasmid expressing a Pa14 CRISPR transcript. Csy4 was affinity purified; co-purifying RNA was extracted and analyzed by denaturing PAGE and staining with SYBR Gold. The ^~19-nucleotide RNA corresponds to a protected fragment of the CRISPR repeat.

Fig. 2. The crystal structure of Csy4 bound to RNA substrate

(A) Front and back views of the complex. Csy4 is colored in blue and the RNA backbone is colored in orange. (B) Csy4 is shown as a surface representation colored according to electrostatic potential. The RNA is shown in ribbon representation with the phosphate backbone colored in orange. The Csy4-RNA complex is shown in the same orientation as in the right panel of (A). (C) Magnified view of the interactions between Csy4 and the major groove of the RNA hairpin. Hydrogen bonding is depicted with dashed lines. (D) Expanded view of the interactions between the arginine-rich helix α3 (blue) and the RNA phosphate backbone (shown in stick format, orange).

Fig. 3. Functional analysis of catalytic residues in Csy4

(A) Detailed view of the catalytic center. Only the phosphate group of nucleotide C21 (the scissile phosphate, indicated with an asterisk) is visible in electron density maps. Strictly conserved residues found in the proximity of the scissile phosphate are shown in stick format. The distance between the hydroxyl group of Ser 148 and the 2’ ribose carbon of G20 is indicated with an arrow. (B) Cleavage activity of Csy4. Wild-type (WT) Csy4 and a series of single point mutants were incubated with in vitro transcribed pre-crRNA for 5 minutes at 25°C. Products were acid phenol-chloroform extracted, resolved by denaturing PAGE and visualized by staining with SYBR Gold.