Molecular determinants for CRISPR RNA maturation in the Cas10-Csm complex and roles for non-Cas nucleases

Abstract

CRISPR–Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a prokaryotic immune system that destroys foreign nucleic acids in a sequence-specific manner using Cas nucleases guided by short RNAs (crRNAs). Staphylococcus epidermidis harbours a Type III-A CRISPR–Cas system that encodes the Cas10–Csm interference complex and crRNAs that are subjected to multiple processing steps. The final step, called maturation, involves a concerted effort between Csm3, a ruler protein in Cas10–Csm that measures six-nucleotide increments, and the activity of a nuclease(s) that remains unknown. Here, we elucidate the contributions of the Cas10–Csm complex toward maturation and explore roles of non-Cas nucleases in this process. Using genetic and biochemical approaches, we show that charged residues in Csm3 facilitate its self-assembly and dictate the extent of maturation cleavage. Additionally, acidic residues in Csm5 are required for efficient maturation, but recombinant Csm5 fails to cleave crRNAs in vitro. However, we detected cellular nucleases that co-purify with Cas10–Csm, and show that Csm5 regulates their activities through distinct mechanisms. Altogether, our results support roles for non-Cas nuclease(s) during crRNA maturation and establish a link between Type III-A CRISPR–Cas immunity and central nucleic acid metabolism.

Figure 1.

CRISPR RNA maturation in a Type III-A CRISPR–Cas system. (A) Illustration of the CRISPR–Cas locus in S. epidermidis RP62a. This system contains four direct repeats (white squares), three spacers (coloured squares), and nine CRISPR-associated (cas and csm) genes. (B) Primary processing of precursor crRNAs is catalysed by Cas6, which cleaves within repeat sequences to yield 71 nucleotide intermediates. (C) The final maturation step involves Csm3, which acts as a ruler that protects 6-nucleotide segments, and the activity of an unknown nuclease that cleaves the unprotected portion of the 3΄ end. These combined activities yield a collection of mature crRNAs (mostly 31–43 nucleotide lengths) that share a uniform 5΄-tag derived from repeat sequences, and variable 3΄-ends that vary by 6-nucleotide increments. (D) A schematic of the Cas10–Csm complex, illustrating the predicted organization of the subunits relative to the crRNA. (E) Csm2, Csm3 and Csm5 are each essential for the maturation process. Csm2 is the small subunit specific to Type III-A systems with unknown function, while Csm3 and Csm5 belong to the Repeat-associated mysterious protein (RAMP) superfamily that possess conserved glycine-rich regions predicted to bind RNA. Conserved residues throughout these proteins that have been explored in previous studies (I) and this study (*) are indicated. See Supplementary Figure S1 for a more detailed depiction of this information. RRM, RNA Recognition Motif.

Figure 2.

Charged residues mediate Csm3 self-interactions and assemble the maturation ruler. (A) Cas10–Csm complexes containing the indicated Csm3 mutations are shown. Mutations were introduced into pcrispr-cas encoding a 6-His tag on the N-terminus of Csm2. Constructs were expressed in S. epidermidis LM1680, and whole cell lysates were subjected to Ni²⁺ affinity chromatography and a second purification using a biotinylated oligonucleotide antisense to spc1 crRNAs. Complexes were resolved and visualized using SDS-PAGE and Coomassie G-250 staining. Shown is a representative of at least three independent trials. (B) RNA was extracted from each complex shown in panel A, radiolabeled on the 5΄-end, and resolved using denaturing PAGE. (C) Purified recombinant Csm3 and mutant variants resolved by SDS-PAGE and visualized using Coomassie G-250 staining are shown. (D) Csm3 and mutant variants (10 pmol each) were resolved alongside the NativeMark unstained protein standard (Thermo Fisher Scientific) by blue native PAGE in which the cathode buffer contained 0.025% Coomassie G-250. Proteins were visualized using Coomassie G-250 staining. Shown is a representative of at least three independent trials. (E) Homology model of a Csm3 oligomer. The Csm3 structure was derived from the high resolution crystal structure of M. kandleri Csm3 (39), and then docked into the A. fulgidus Cmr4 backbone of a Type III-B complex (34). K4 and D179 residues are shown in space-fill.

Figure 3.

Acidic amino acids in Csm5 are necessary but insufficient to conduct crRNA maturation. (A) Cas10–Csm complexes containing the indicated Csm5 mutations are shown. Mutations were introduced into pcrispr-cas encoding a 6-His tag on the N-terminus of Csm2. Constructs were expressed in S. epidermidis LM1680, and whole cell lysates were subjected to Ni²⁺ affinity chromatography and a second purification using a biotinylated oligonucleotide antisense to spc1 crRNAs. Complexes were resolved and visualized using SDS-PAGE followed by Coomassie G-250 staining. The dotted line separates non-contiguous lanes within the same gel. (B) RNA was extracted from each complex in panel A, radiolabeled on the 5΄-end, and resolved using denaturing PAGE. (C) Purified recombinant Csm5 resolved by SDS-PAGE and visualized using Coomassie G-250 staining is shown. (D) 71-nucleotide intermediate crRNAs were extracted from Cas10–Csm/ΔCsm5 complexes (shown in panel A) and used as substrates in a nuclease assay containing purified Csm5 (8 pmol) and EDTA or various metals as indicated. The reaction was allowed to proceed at 37°C for 10 min. RNAs were resolved by denaturing PAGE. Shown is a representative of at least five independent trials with three different Csm5 preparations. (E) The 31-nucleotide spc1 crRNA substrate used for Csm5 binding and nuclease assays is shown. The 5΄ tag sequence is boxed in white, and spacer 1-derived sequence is shaded. The asterisk indicates the radiolabel on the 5΄-end. (F) A double-filter binding assay is shown. Increasing amounts of Csm5 (0, 0.5, 1, 2, 8 and 32 pmol) and trace amounts of 5΄-end labeled RNA substrate (panel E) were combined and applied to wells in triplicate. Csm5-bound probe (upper membrane) and free RNA (lower membrane) are indicated. (G) Quantification of the double-filter assay showing the fraction of RNA bound as an average of triplicate measurements (±S.D.)

Figure 4.

Csm5 differentially modulates the activity of cellular nucleases. (A) Purified recombinant Cbf1, PNPase and RNase R resolved by SDS-PAGE and visualized using Coomassie G-250 staining are shown. (B and C) Csm5 protection against Cbf1 (B) and RNase R (C) activity is shown. 5΄-end labelled 31-nucleotide spc1 crRNA substrate (Figure 3E) was pre-incubated with increasing amounts of Csm5 (0, 1, 2, 4 and 8 pmol) before nuclease addition (1 pmol each). The reaction mixture was incubated at 37°C for 10 min. RNAs were resolved using denaturing PAGE. Full-length substrate (arrow), degradation intermediates (asterisks, *), and fully degraded substrate (brackets) are indicated. (D) Quantification of the Csm5 protection assays shown in panels B and C. Percent substrate protected was calculated as follows: ((band intensities of full length substrate + degradation intermediates)/total substrate added)) × 100. Shown is the average (±S.D.) of three independent trials. (E) PNPase (1 pmol) was combined with a radiolabeled 31 nucleotide spc1 crRNA substrate and Csm5 (4 pmols) where indicated. The reaction mixture was incubated at 37°C for increasing time points (0, 2, 5, or 10 minutes.) RNAs were resolved using denaturing PAGE. (F) The 5΄-end labelled 22-nucleotide DNA substrate used in nuclease assays. (G) PNPase or Cbf1 (1 pmol each) were combined with the DNA substrate (panel F) in the presence of Csm5 (4 pmol) where indicated. The reaction mixture was incubated at 37°C for 10 min, and DNAs were resolved using denaturing PAGE. All gel images are a representative of at least three independent trials.

Figure 5.

Csm5 physically interacts with PNPase. (A) Abbreviated experimental flow of a Ni²⁺ affinity pulldown assay, in which His₁₀Smt3 tagged Csm5 (Csm5-Smt3, ‘input 1’, 1 nmol) is loaded onto a column containing Ni²⁺-agarose beads and washed. Untagged PNPase (‘input 2’, 0.7 nmol) is then passed through the column, and anything unbound is thoroughly washed. Proteins retained in the column are eluted using 500 mM imidazole. Abbreviations are as follows: FT, flow-through; W, wash; imid., imidazole. (B) Samples from the Csm5–Smt3 (input 1), untagged PNPase (input 2), and final elution were resolved using SDS-PAGE. The final elution from a negative control is also shown in which dialysis buffer was used as input 1. See Supplementary Figure S5 for a detailed description of the experiment and all samples collected during the experiment. The dotted line separates non-contiguous lanes within the same gel. Shown is a representative of four independent trials. (C) Csm5 and PNPase (100 pmol each) were resolved on a 5% vertical native gel alone or in combination (1:1 ratio). The position of (+) and (-) electrodes are indicated. (D) Numbered bands shown in panel C were excised from the native gel and the proteins within were resolved using denaturing SDS-PAGE. Arrowheads mark the top of each excised band and indicate band orientation when run in second dimension. Shown is a representative of three independent trials. Proteins were visualized with Coomassie G-250.

Figure 6.

A model for crRNA maturation. Maturation occurs during Cas10–Csm complex assembly with intermediate crRNAs. Charged residues in Csm3 promote its self-interactions as it polymerizes on the crRNA. Csm5 facilitates crRNA maturation by recruiting PNPase to the 3΄-end of the crRNA. Once it joins the complex, Csm5 shields crRNAs from further nonspecific degradation by PNPase and other cellular nucleases.