RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus

Abstract

CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1-Csm5) with an uneven stoichiometry and a single crRNA of variable size (35-53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence.

Figure 1. Schematic Representation of CRISPR Arrays and cas Genes on the Chromosome and Plasmid pTT27 of T. thermophilus HB8

CRISPR arrays (1–12) are indicated in different grayscales, depending on the repeat type (I, II, or III). Cas (-related) genes belonging to a particular CRISPR-Cas subtype are colored in green (subtype III-A), blue (subtype III-B), or yellow (subtype I-E). Additional cas genes are indicated in white. For each of these CRISPR arrays, the bottom panel summarized the genomic location, the consensus repeat sequence, repeat type, and the number of spacers. The 5′ tag sequences, as found by our RNA-seq analysis, are underlined. The figure and legend are adapted from Staals et al. (2013).

Figure 2. Purification of the Native T. thermophilus Type III-A Csm Complex

(A) SDS-PAGE analysis of the T. thermophilus Csm complex. A representative sample of the purified protein (2 μg) was analyzed on a 15% polyacrylamide gel (lane 2), followed by staining with Coomassie Brilliant Blue R-250. Each subunit is indicated. The Csm5 has a (His)₆-tag at its C terminus. Lane 1, molecular-mass markers. (B) BN-PAGE analysis of the T. thermophilus Csm complex. Two μg of the representative sample was analyzed on a 4%–16% linear polyacrylamide gradient gel in the presence of 0.02% Coomassie Brilliant Blue G-250 (lane 2). Lane 1, molecular-mass markers. Protein concentration used was determined by the Bradford method (Bradford, 1976), using bovine serum albumin as a standard.

Figure 3. RNA-Seq Analysis of TtCsm-Bound crRNAs

(A) crRNAs were isolated by phenol-chloroform-isoamyl alcohol extraction and analyzed by denaturing PAGE (20% AA, 7M urea). Discontinuous gel lanes are indicated by dashed lines. (B) Histogram of the size-distributions of the Csm-bound crRNAs. (C) Histogram of the distribution of the Csm-bound crRNAs over the 11 CRISPR arrays. (D) Mapping of the Csm-bound crRNAs on CRISPR4. Overview of all mapped crRNAs and comparison of the crRNA content of the TtCsm and TtCmr complexes are provided in Figure S1.

Figure 4. Subunit Composition of TtCsm

(A) Native nanoelectrospray ionization mass spectrum of the native TtCsm complex. Two main well-resolved charge state distributions are present at high m/z values, corresponding to complexes of 427 kDa (blue) and 382 kDa (red). (B) TtCsm (sub)complexes analyzed by native mass spectrometry. The subcomplexes were formed after in-solution dissociation with 30% DMSO v/v or 175 mM ammonium acetate acidified with acetic acid (pH 3.6–4). More in-depth calculations of the different (sub)complexes can be found in Table S1 and Table S2.

Figure 5. In Vitro Activity Assays with the TtCsm Complex

(A) A 5′-labeled ssRNA target complementary to crRNA 4.5 was incubated with different concentration of the purified, endogenous TtCsm complex in a buffer containing 2 mM Mg²⁺. Samples were analyzed by denaturing PAGE (20% AA, 7 M urea), followed by phosphorimaging. (B) The ssRNA target was incubated with 100 nM of the endogenous Csm or Cmr complex for the indicated amount of time. OH, alkaline hydrolysis ladder of the 50 nt RNA target. (C) RNA targets (complementary to crRNA 4.5) with base-pair-disrupting mutations at the indicated positions (also see Figure 6A) were incubated in the absence (“−”) or presence (“+”) of TtCsm. In order to visualize more (transient) degradation products, the assay was performed with a lower (10 μM) Mg²⁺ concentration. “WT” refers to the unmodified “wild-type” target RNA. (D) RNA targets (complementary to crRNA 4.5) with mutated, base-pair-disrupting regions at the indicated positions (also see Figure 6A) were incubated in the absence (“−”) or presence (“+”) of TtCsm in a buffer containing 10 μM Mg²⁺. Additional Csm activity assays with different RNA or DNA substrates and different cofactors are provided in Figure S3. Discontinuous gel lanes are indicated by the dashed line.

Figure 6. Model of Cleavage Activity of the TtCsm Complex

(A) Schematic representation of the cleavage activity of the TtCsm complex. (B) Schematic representation of the impact of base-pair-disrupting mutations in regions of the target RNA on activity (also see Figure 5D). Cleavages observed in this study are indicated by dotted lines. Skipped cleavage sites are indicated with red crosses. Nucleotides in red indicate repeat-derived sequences of the crRNA.

Figure 7. Molecular Architecture of the T. thermophilus Csm Complex

(A) Raw micrograph of negatively stained TtCsm complexes. Scale bar, 100 nm. (B) Representative reference-free 2D class averages of TtCsm complexes. The width of the boxes is ~400 Å. (C) Working segmentation of the TtCsm complex reconstruction at 17 Å resolution highlighting the “sea worm” architecture. Segmented regions are colored and labeled as Csm1 (purple), Csm2 (red), Csm3 (alternating light blue and gray), Csm4 (green), and Csm5 (orange).