A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.

FIGURE 1.

Cleavage of ssRNAs by CAS2 proteins SSO1404, SSO8090, TM1796, NE0845, AF1876, and MTH1083. Autoradiograph of the 15% PAA, 8 m urea gels showing the hydrolysis of RNA5 (A), RNA6 (B), and RNA14 (C) by the indicated CAS2 proteins. RNA substrates (0.1 μm) were incubated at 37 °C for 45 min with the CAS2 protein (50 μg/ml) in the presence of 50 mm Tris-HCl (pH 8.5), 5 mm MgCl₂, and 100 mm KCl. Reaction products were separated on a 15% PAAG, 8 m urea gel as described under “Experimental Procedures.” Lane C represents the RNA sample incubated in the absence of protein; lane M shows the 5′-³²P-labeled marker oligonucleotides.

FIGURE 2.

Reaction requirements and RNA cleavage products of SSO1404. Autoradiographs of 15% PAA, 8 m urea gels showing the effect of pH (A) or various metal cations (B) on the cleavage of RNA5 by SSO1404 (30 μg/ml). A, 5′-³²P-labeled RNA5 (0.1 μm) was incubated with SSO1404 at 37 °C for 10 min in a reaction mixture containing 50 mm buffer with the indicated pH (described under “Experimental Procedures”), 5 mm MgCl₂, 100 mm KCl, and 1 mm dithiothreitol. B, 5′-³²P-labeled RNA5 (0.5 μm) was incubated with SSO1404 in the absence or presence of 5 mm MgCl₂, 5 mm MnCl₂, 100 mm NaCl, or 100 mm KCl. C, analysis of the RNA5 cleavage products generated by SSO1404: lane 1, reaction mixture without the addition of PNK; lane 2, reaction mixture treated with PNK (5′-end labeling reaction); lane 3, reaction mixture treated with PNK (5′-phosphate exchange reaction). Lane C represents the RNA sample incubated in the absence of protein; lane M shows the 5′-³²P-labeled marker oligonucleotides. Reaction products were processed and visualized as described under “Experimental Procedures.” The position of the main RNA5 cleavage product (U₉-U₁₀) is shown on the right. A T1 ladder represents the products of RNA5 cleavage by RNase T1.

FIGURE 3.

RNase activity of SSO1404 with CRISPR-related substrates. A, cleavage of the 5′-fragment (270 nt) of the S. solfataricus CRISPR cluster-2 containing the cluster-2 upstream region, repeat-1, spacer-1, repeat-2, and a 27-nt fragment of spacer-2. Autoradiograph of the 8% PAA, 8 m urea gel showing the cleavage of the uniformly labeled [³²P]RNA (0.1 μm) by SSO1404 (30 μg/ml). RNA was incubated in the absence of SSO1404 (30 min, lane 1) or in the presence of SSO1404 for (15 min, lane 2) or (30 min, lane 3). B, RNA1 to RNA5; C, RNA6 to RNA9; D, RNA10 to RNA12. The right part of the of B-D shows predicted secondary structures and the positions of the SSO1404 cleavage sites in selected RNA substrates. Im, imidazole.

FIGURE 4.

Ribonuclease activity of SSO1404 against a series of the truncated RNA5 substrates. A, autoradiograph of the 15% PAA, 8 m urea gel showing the activity of SSO1404 against the RNA5 substrate and its five truncated derivatives (the substrate lengths are shown on the right). B, sequences of the substrates used in the experiment and the positions of the cleavage sites of SSO1404. RNA substrates (0.1 μm) were incubated for 15 min (37 °C) in the absence (lanes 1, 3, 5, 7, 9, and 11) or in the presence (lanes 2, 4, 6, 8, 10, and 12) of SSO1404 (30 μg/ml). Reaction products were processed and visualized as described under “Experimental Procedures.” Lanes T1 and Im represent the products of the RNA5 cleavage by RNase T1 and 2 m imidazole (pH 7.0), respectively.

FIGURE 5.

Structures of SSO1404 and other CAS2 proteins. A, SSO1404 dimer. One of the two monomers is colored under “rainbow” representation (blue at the N terminus to red at the C terminus). B, SSO1404 monomer. C, TT1823 monomer (Protein Data Bank code 1zpw). D, PF1117 monomer (Protein Data Bank code 2i0x). All of the polypeptides are shown in equivalent orientations and are colored under rainbow representation.

FIGURE 6.

Potential active site of SSO1404. A, surface representation (two orientations) of the SSO1404 dimer showing the potential active site: the deep cavity between two monomers harboring two conserved Asp-10 (colored red). One of the monomers is colored in cyan, and the other is in green. Residues colored orange (Gln-63, Glu-64, and Asp-65) are located on the two α2-β4 loops, which are predicted to be involved in RNA binding. Other conserved residues important for activity are shown in yellow (Tyr-9, Arg-17, Arg-19, Arg-31, and Phe-37). B, stereo view of the SSO1404 dimer showing the positions of the residues important for activity (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37). One of the monomers is colored in cyan, and the other is in green. The orientation of the SSO1404 dimer in B corresponds to the right model of A. Note the proximity of the two principal catalytic Asp-10 residues in the dimer (6.5 Å).

FIGURE 7.

Structure-based sequence alignment of SSO1404 and selected CAS2 proteins. Residues conserved in all CAS2 proteins are highlighted in black, highly conserved residues in dark gray, and similar residues in light gray. The secondary structure elements derived from the structure of SSO1404 (Protein Data Bank code 2i8e) are shown above the alignment. The asterisks designate the residues important for the catalytic activity of SSO1404. The compared proteins are as follows:SSO1404(Q97YC2), SSO8090(Q97Y85), TT1823 from T. thermophilus(Q746F4), PF1117 from P. furiosus (Q8U1T8), AF1876 from A. fulgidus (O28403), MTH1083 from M. thermoautotrophicum (O27155), TM1796 from T. maritima (Q9X2B6), and NE0845 from N. europaea (Q82W51). The sequences were aligned using both sequence and structural information by 3DCOFFEE (63), and the figure was generated using TEXSHADE (64).

FIGURE 8.

Site-directed mutagenesis of SSO1404, RNase activity of purified mutant proteins with the RNA5 substrate. Autoradiograph of the 15% PAA, 8 m urea gel showing the cleavage of 5-³²P-labeled RNA5 (0.1 μm) by purified proteins (30 μg/ml). Reaction mixtures were incubated at 37 °C for 10 min, and the reaction products were processed as described under “Experimental Procedures.” Lane C represents the RNA5 sample incubated without protein addition.