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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Structure. Author manuscript; available in PMC 2017 April 5.
Published in final edited form as:
PMCID: PMC4823167
NIHMSID: NIHMS764199

A Non Stem-loop CRISPR RNA Is Processed by Dual Binding Cas6

Abstract

A subclass of recently discovered CRISPR repeat RNA in bacteria contain minimally recognizable structural features that facilitate an unknown mechanism of recognition and processing by the Cas6 family of endoribonucleases. Cocrystal structures of Cas6 from Methanococcus maripaludis (MmCas6b) bound with its repeat RNA revealed a dual-site binding structure and a cleavage site conformation poised for phosphodiester bond breakage. Two non-interacting MmCas6b bind to two separate AAYAA motifs within the same repeat, one distal and one adjacent to the cleavage site. This bound structure potentially competes with a stable but nonproductive RNA structure. At the cleavage site, MmCas6b supplies a base pair mimic to stabilize a short two base-pair stem immediately upstream of the scissile phosphate. Complementary biochemical analyses support the dual-AAYAA binding model and a critical role of the protein-RNA base pair mimic. Our results reveal a previously unknown method of processing non stem-loop CRISPR RNA by Cas6.

Keywords: Protein-RNA interactions, endoribonucleases, CRISPR RNA, crystal structures

Graphical abstract

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INTRODUCTION

Ribonucleic acids (RNA) possess an inherent structural flexibility that enables them to serve in a broad range of biological activities. Endoribonucleases are protein enzymes that cleave RNA, often at a specific phosphodiester bond, to yield functional forms of RNA. The principle of how endoribonucleases interact with and cleave intrinsically flexible RNA substrates remains only partially understood owing in part to the lack of three-dimensional structures of endoribonucleases bound with their RNA substrates.

Cas6 superfamily endoribonucleases function in a small RNA-guided immunity pathway conferred by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated proteins (Cas proteins) in prokaryotes (Hochstrasser and Doudna, 2015; Li, 2015). The CRISPR loci are segments of identical repeat sequences (repeats) interspersed by unique spacer sequences (spacers) (Barrangou and Marraffini, 2014; Gasiunas et al., 2014; Reeks et al., 2013; van der Oost et al., 2014; Wiedenheft et al., 2012). The repeat-spacer array encodes RNA substrates for Cas6. Cas6 recognizes features exclusively within the repeat and cleaves, in almost all cases, 8 nucleotides away from the 3′ end of the repeat, resulting in the spacer RNA flanked by repeat sequences at both ends (CRISPR RNA or crRNA) (Hochstrasser and Doudna, 2015; Li, 2015). Processed crRNA function as guides to the interference ribonucleoprotein particles (crRNPs) in identifying foreign nucleic acids (e.g. viral genomes or conjugative plasmids) to be degraded by Cas proteins (Barrangou and Marraffini, 2014; Gasiunas et al., 2014; Reeks et al., 2013; van der Oost et al., 2014; Wiedenheft et al., 2012).

The letter “P” in the CRISPR acronym reflects the “Palindromic” feature detected in slightly more than 50% of the known repeat sequences (Kunin et al., 2007; Lange et al., 2013). The palindrome in CRISPR repeats results in stem-loop structures in their encoded RNA that are key features recognized by a number of known Cas6 endoribonucleases (Hochstrasser and Doudna, 2015; Li, 2015). In these cases, the Cas6 endoribonucleases depend on the hallmark stem-loop feature and cleave at the base of the stem. The 3′ cleavage product is released after cleavage but the 5′ cleavage product, which includes the stem-loop, remains tightly bound to Cas6 that ultimately becomes part of the downstream interference crRNPs (Hochstrasser and Doudna, 2015; Li, 2015). Thus, the presence of the palindromic feature in repeats is indicative of the stem-loop of the bound RNA substrate and therefore, their mode of processing by Cas6.

Significantly, nearly half of the known repeats exhibit weak or no palindromic features, making prediction of the RNA processing mechanism difficult (Kunin et al., 2007; Lange et al., 2013). Most of these repeat RNAs are also recognized and cleaved by Cas6 from the same 8 nt distance to the 3′ end of the repeat. The Cas6 proteins associated with these repeat RNAs are predicted to have similar three-dimensional structures as those associated with stem-loop RNAs. Biochemical studies showed that these Cas6 proteins tend to have weaker binding affinities for their 5′ cleavage product and therefore do not associate with the downstream interference crRNPs (Hochstrasser and Doudna, 2015; Li, 2015). In contrast to the well-accepted processing model for the stem-loop repeat RNA, our understanding of how non stem-loop repeat RNA are recognized by Cas6 is limited due to the large variations in RNA structures and currently inconsistent RNA binding results. Two examples of Cas6 binding to non stem-loop repeat RNA have been studied. A short Sulfolobus solfataricus (Ss) repeat RNA is processed through stabilization of a three base pair stem-loop by SsCas6 (Shao and Li, 2013), suggesting an intrinsic ability of Cas6 in reinforcing a stem-loop near the cleavage site. However, Pyrococcus furiosus (Pf) repeat RNA is specifically recognized by its 5′ end as well as its cleavage site, leading to the wraparound model in which the repeat RNA anchors its 5′ end on one side and the cleavage site on the other of a single Cas6 subunit (Wang et al., 2011). A thorough understanding of CRISPR RNA processing is not possible without a comprehensive knowledge associated with a wide range of repeat RNAs.

Formation of the stem immediately upstream of the cleavage site facilitates phosphodiester bond breakage catalyzed by Cas6. The Cas6 endoribonucleases known so far employ an RNase A-like cleavage mechanism (Cuchillo et al., 2011) in which a general base extract the proton from the 2′-hydroxyl group immediately upstream of the scissile phosphate and a general acid donates a proton to the 5′ leaving oxygen associated with the scissile phosphate (Hochstrasser and Doudna, 2015; Li, 2015). A positively charged residue near the scissile phosphate stabilizes the developing negative charge of the transition state. The SN2 reaction requires an alignment of the 2′-hydroxyl oxygen, the scissile phosphate and the 5′ leaving oxygen of the RNA (inline conformation), which is incompatible with the A-form RNA structure. On the other hand, the base of a stem-loop RNA is ideal for inline conformation formation due to splaying of its downstream nucleotide. It is thus predicted that the non-stem loop RNA substrates for Cas6 may also form analogous stem loops at the expense of Cas6 binding (Li, 2015; Shao and Li, 2013). However, experimental observations of non-stem loop RNA bound with Cas6 remain scarce, raising the question of how Cas6 achieve specific recognition of the non stem-loop repeat RNAs.

Methanococcus maripaludis C5 (Mm) contains a type I-B Cas6, MmCas6b (MmarC5_0767), responsible for processing crRNA (Richter et al., 2013; Richter et al., 2012). The RNA substrate of MmCas6b, the NC_009135 repeat, contains 37 nucleotides with a calculated minimum free energy stem-loop structure (13–28) one nucleotide upstream of the cleavage site at position 29 (Figure 1). To understand the biochemical mechanism of Cas6b, we determined two cocrystal structures of MmCas6b bound with its RNA substrate analogs. The complex structure with a non-cleavable (2′-deoxy modification at position 29), near full-length, RNA substrate (d31mer) at 3.0 Å resolution reveals two unexpected dual binding motifs optimal for cleavage site recognition. The complex structure with a minimal recognition motif RNA (14mer) at 3.5 Å resolution reveals negligible structural changes in the active site despite the lower stability of the complex, suggesting a role of the distal minor motif in aiding substrate recognition rather than catalysis. Dual recognition by Cas6 reconciles the previous inconsistent binding data among Cas6 proteins and provides a strategy for Cas6 endoribonucleases to process CRISPR repeats lacking stable stem-loop structures.

Figure 1
Overview of the MmCas6b-RNA complex (d31mer) structure

RESULTS

MmCas6b recognizes dual RNA motifs

Although free energy minimization predicts a stem-loop structure for the NC_009135 repeat RNA in isolation (Figure 1A), it forms two recognition motifs (I & II) when bound to MmCas6b (Figure 1A & 1B). One of the two bound MmCas6b (subunit A) binds at the minor recognition site comprising nucleotides 7–12 (motif I) and the other (subunit B) binds at the major recognition site comprising nucleotides 16–30 including the cleavage site between nucleotides 29 and 30 (motif II) (Figure 1A & 1B). The two motifs are linked by three extended nucleotides 13–15 (~ 40 Å), which results in few contacts between the two bound MmCas6b subunits with only 235 Å2 buried solvent accessible surface area.

To assess the impact of the distal motif I on the structure of the MmCas6b-RNA complex, we also analyzed a crystal structure of MmCas6b bound with a 14mer RNA containing only motif II nucleotides excluding the cleavage site (nucleotides 16 - 29) (Figure 2). MmCas6b binds to one 14mer molecule as an isologous homodimer where one subunit contains no bound RNA (Figure 2A). The homodimer buries a 598 Å2 solvent accessible area averaged among the three complexes in the asymmetric unit, suggesting a weak but discernible dimerization interaction. The moderate interface of the 14mer-bound dimer observed in the crystal structure likely explains the small proportion of dimer revealed in a gel filtration experiment with the RNA-free MmCas6b (Figure S1). The 14mer-bound dimer is unrelated to the two subunits bound with the d31mer RNA that interact only with the d31mer and not with each other. To compare MmCas6b structures when free and bound with different RNA, we superimposed the structures of the four available MmCas6b subunits (two from each structure). The 14mer-bound subunit is nearly identical to that bound to the motif II in the d31mer complex (Figure S2). Thus, motif I has no significant impact on the structure of the bound motif II. The RNA-free subunit is also in general similar to the motif II-bound subunit except that its α1 and part of α2 helices are disordered (Figure 2B). Interestingly, the 14mer- or the motif II-bound subunit differs from the motif I-bound subunit in α1 and α2 orientations (Figure 2B), suggesting that the short stem in motif II induces the conformational changes in α1 and α2 that are likely required for catalysis.

Figure 2
Structure of the MmCas6b-14mer RNA complex and structural comparison

To assess how protein:RNA stoichiometry depends on the two motifs in solution, we carried out electrophoretic mobility shift assays of MmCas6b with the repeat RNA in the presence (d37mer) and absence (14mer) of motif I. The binding of the d37mer RNA substrate showed a single retarded species at low MmCas6b concentrations that was further retarded as additional MmCas6b was added (Figure 3A & 3B). A similar stepwise shift of the wild-type repeat RNA was previously observed (14). These observations suggest a sequential binding of motif II and motif I by MmCas6b. In contrast, binding of the 14mer RNA resulted in a single retarded species for all MmCas6b concentrations (Figure 3A & 3B), consistent with the presence of only a single binding site in this RNA. Interestingly, the retarded species of the motif I-lacking 14mer migrated slower than that of the lower shifted species of the d37mer. It is plausible that the additional retardation represents the cooperative dimerization of MmCas6b as observed for the MmCas6b-14mer complex.

Figure 3
RNA binding and cleavage assay results

Measurement of binding constants revealed notable difference between MmCas6b binding to the dual- and the single-motif RNA oligomers. Fitting filter binding isotherms to a Hill equation showed ~40x increase in Kd when binding the 14mer than the d31mer (Figure 3A & 3B), suggesting an impact of motif I on MmCas6b binding thermodynamics. In contrast, removal of the last eight nucleotides from the 3′ end (23mer) had only a minor impact on the binding constant (Figure 3B). The non-hyperbolic binding isotherms for both RNA oligomers is consistent with the observed 2:1 protein:RNA stoichiometry ratio in both the 31mer and the 14mer complexes. Although a truncated RNA lacking the AAUAA of motif I was cleaved by MmCas6b (25mer), further truncation of two nucleotides towards motif II (18mer) severely impacted cleavage by MmCas6b (Figure 3C). Taken together, these results suggest that the motif I greatly facilitates but is not required for cleavage at motif II by MmCas6b.

RNA recognition by MmCas6b

Motif II of the repeat RNA is the major site recognized by MmCas6b. It comprises a two base pair stem (G16-C29/C17-G28) and an adenine-rich loop (A18-A27). The major groove of the short stem faces the interior of MmCas6b, and together with the major groove edge of U15, forms a polar interface with the protein at which Arg206 of the G-loop interacts directly with the N4 atom of C29 and, through the carbonyl oxygen of Leu124, with U15 (Figure 4). Other residues do not form base-specific hydrogen bonds but pack around the two base pairs. These include Gln183 of loop β6-β7, Asn39, His40, and Phe45 of Loop α1-β2, and Asn207 and Ser209 of the G-loop (Figure 4). The adenine-rich loop wraps mostly around the α3 and partially around the αA helices and contacts several asparagine residues via its curved phosphosugar backbone (Figures 1 & 4). Only A26 and A27 of the loop are involved in base interactions with the main chain atoms of the MmCas6b residues (Figure 4). Motif I comprises a similar adenine-rich loop and its A8AUAA12 loop is nearly identical to A23ACAA27. Consequently, the two motifs share the AAYAA (Y=C, U) sequence and use the same surface of the subunit A to interact with the adenine-rich loop (Figure 4).

Figure 4
Details of MmCas6b-RNA interactions. Four regions of the protein-RNA interactions were highlighted for detailed descriptions (panels A-D). Note that motifs I and II share similar interactions involving the AAYAA (Y=U, C) sequence

The most unusual feature of MmCas6b-RNA interaction involves a protein-RNA base pair mimic between Tyr47 and A18. The aromatic ring of Tyr47 inserts into the RNA stem and pairs with the minor groove edge of A18, forming an RNA base pair mimic (Figure 4). The Tyr47-A18 base pair mimicry is enabled by unstacking and splaying of the A-rich loop that gives away space for Tyr47 to stack on top of the two base pair stem. In addition, A19 stacks on the Tyr47-A18 pair while its base interacts with the major groove edge of A21. This mode of MmCas6b-RNA interaction creates a hybrid stem structure upstream of the cleavage site.

Consistent with the observed structural features, mutation of G16 to C greatly impaired the cleavage and double mutation G16C/C25G abolished the cleavage activity (Figure 3D). Furthermore, Tyr47Ala mutation greatly reduced cleavage activity (Richter et al., 2012). In contrast, mutation U15A did not appreciably affect the cleavage activity (Figure 3D). To further confirm the spatial positioning of U15, we performed protein-RNA UV-crosslinking followed by mass spectrometry analysis. The base of U15 was indeed observed to cross-link with Met185 after UV-irradiation of the Cas6b-repeat RNA complex in solution (Figure 5), providing a convincing evidence for the observed binding mode of U15 (Figure 4B).

Figure 5
MS and MS/MS spectra of Cas6b peptide NQNMoxVGFR cross-linked to RNA oligonucleotide UUGC-PO3. The cross-linked peptide with its cross-linked RNA oligonucleotide was identified and sequenced with the help of RNPxl software applied on MS raw data (Kramer ...

The active site structure of MmCas6b

Phosphodiester bond cleavage by MmCas6b takes place between C29 and A30. The nucleotide A30 splays away from the helical track of the stem towards α1 helix (Figure 6A). The rotation of A30 around the scissile phosphate bond is critical to formation of the conformation favorable for catalysis. Due to the use of 2′-deoxy modification of C29 in crystallization, the 2′-hydroxyl oxygen is absent from our structure. However, if it were present, the three reactive atoms (2′-hydroxyl oxygen, phosphate, and 5′-oxygen) would form the inline conformation (Figure 6A). Two histidine residues (His38 and His40) and four positively charged residues (Arg24, Lys29, Lys181, and Arg210) surround the cleavage site (Figure 6A). His40 is in a position to deprotonate the 2′-hydoxyl group and Arg24 or Arg210 is in a position to donate proton to the leaving 5′-oxygen. Lys181 or Arg210 probably stabilizes the negative charge of the transition state. The imidazole ring of His38 interacts with the splayed A30 nucleotide (Figure 6A). Consistently, mutations of His40 and His38 both reduced the cleavage activity and their double mutation abolished cleavage (Richter et al., 2012). Interestingly, mutation of Arg24 or Lys29 to alanine did not reduce RNA cleavage activity (Richter et al., 2012), suggesting the redundant roles of these residues in RNA cleavage. Alternatively, MmCas6b relies primarily on the general base His40 and less so on a general acid for protonating the leaving oxygen.

Figure 6
Structure of the MmCas6b active site (A) and the model of dual binding processing by Cas6 (B)

DISCUSSION

We determined the X-ray crystal structure of a Cas6 crRNA processing endonuclease (MmCas6b) bound with its RNA substrate analogs. MmCas6b cleaves within the repeat region of the precursor crRNA 8 nts from its 3′ end. The structure of MmCas6b-RNA complex revealed a surprising secondary structure of the repeat RNA that is stabilized by a base pair mimic and a second MmCas6b subunit bound to a minor recognition site. This secondary structure permits proper recognition of the cleavage site in spite of a competing, potentially more stable, structure. The two base pair stem immediately upstream of the cleavage site facilitates the inline conformation of the scissile phosphate bond required for cleavage. This mode of Cas6-RNA recognition illustrates the power of protein enzymes in shaping structurally flexible RNA for catalysis.

MmCas6b is a member of Cas6 RNA processing endoribonucleases found in bacteria and archaea whose RNA substrates have a wide range of structural features. While recognition and processing of stable stem loop RNA are well understood due to several high resolution structures of Cas6 bound with their respective stem-loop RNA substrates (Hochstrasser and Doudna, 2015; Li, 2015), these processes not clear for repeat RNA lacking stable stem-loops. One method of processing is for Cas6 to impose a stem-loop structure of the RNA that is otherwise unstable in isolation. Sulfolobus solfataricus Cas6 bound with its short repeat RNA (24mer) illustrates this principle (Shao and Li, 2013). However, the extent to which Cas6 stabilizes a short stem loop structure is limited for long repeat RNA containing potentially competing secondary structures. MmCas6b structures reported here provide a second method of recognition in which the free 5′ end of the repeat is stabilized by one Cas6 subunit so the cleavage site can be shaped by another Cas6 subunit (Figure 6B). Although in this case the 5′ distal recognition was shown to be nonessential to cleavage, other Cas6 may have evolved to depend more critically on sites beyond the cleavage site to ensure a catalytically competent conformation at the cleavage site. The added binding site(s) for Cas6 may have an evolutionary advantage in mesophilic organisms where it is difficult to dissolve competing structures in long pre-crRNA transcripts. Pyrococcus furiosus (Pf) Cas6 may ialso adopt this method of recognition with a stringent requirement for the presence of the 5′ terminus of the repeat RNA (Carte et al., 2008). Consistently, crystal structures of PfCas6 and its close homolog Pyrococcus horikoshii (Ph) Cas6 reveal their specific interactions with the 5′ terminus of the repeat RNA (Wang et al., 2011; Wang et al., 2012). The dual binding model allows Cas6 to overcome the challenge presented in the repeat RNA containing competing and non-productive secondary structures and may thus have general applicability in more Cas6 processing systems.

MATERIALS AND METHODS

Protein purification and crystallization

The M. maripaludis cas6 gene, MmarC5_0767, was cloned into the vector pET20b with a C-terminal 6×his affinity tag. The MmCas6b protein was overexpressed in E. coli Rossetta strain and purified by a similar method as that previously described (Richter et al., 2012). Briefly, the log phase E. coli cells were induced by adding IPTG to 0.5 mM and harvested following overnight growth at 18°C. The cell pellets were re-suspended in a lysis buffer (10 mM Tris–HCl pH7.5, 500 mM NaCl, 10% glycerol and 0.5 mM DTT) and disrupted by sonication. The supernatant was loaded onto a Ni2+-NTA affinity column and eluted by the same buffer supplemented with 350 mM imidazole. The elutant was further purified by Superdex 200 gel filtration column equilibrated with 500 mM NaCl, 20 mM Tris-Cl pH7.5, 5% glycerol. The peak fractions corresponding to the monomer species (Figure S1) were pooled and concentrated to 50 mg/mL and stored in −80°C until crystallization.

The RNA oligomers were ordered from Integrated DNA technology (Coralville, IA). MmCas6b and the RNA were combined with 1:1.2 ratio and the complexes were crystallized at 30°C using hanging drop vapor diffusion method by mixing equal volumes of well solution and protein-RNA complexes. MmCas6b with 31mer RNA substrate analog was crystallized in 100 mM KCl, 10mM MgCl2, 50mM sodium cacodylate pH 6.5, 12% PEG4000. Crystals were cryo-protected in a well solution supplemented by 25% glycerol before being flash-frozen in liquid nitrogen. MmCas6b bound with 14mer was crystallized in 20 mM magnesium acetate, 50 mM sodium cacodylate pH 5.5, and 1.7 M ammonium sulfate. Crystals were soaked overnight in the well solution supplemented with 20 mM HgCl2 followed by cryo-protection in well solution supplemented with 25% glycerol before being flash-frozen in liquid nitrogen.

Data Collection and Structure Determination

Single wavelength anomalous x-ray diffraction (SAD) data of the MmCas6b-14mer complex was collected at the mercury absorption edge and processed using HKL2000 (Otwinowski and Minor, 1997), which allowed determination of experimental phases with Phenix.autosol (Adams et al., 2010). Most of the protein residues and the 14mer RNA could be unambiguously built from the solvent flattened density map and the structure was refined with Phenix.refine (Adams et al., 2010). The crystal structure of MmCas6b-d31mer complex was determined by molecular replacement using MmCas6b from the 14mer complex as the probe with Phenix.Phaser (Adams et al., 2010). The structural models were improved by Coot (Emsley et al., 2010) and refined by Phenix.refine (Adams et al., 2010) (Table 1). Throughout refinement of both structures, torsion-angle noncrystallographic symmetry restraints were applied. A small fraction of twinning by merohedry was detected by using the Wilson intensity ratio analysis. Three possible merohedral twin operators (-h,-k,l, h; -h-k,-l; and –k,-h,-l) were identified that resulted in 5%, 6%, and 18% twinning fractions, respectively. The molecular replacement solution was successful without any detwin operation. Similarly, refinement and model building proceeded satisfactorily without detwin. However, inclusion of the (-k, -h, -l) twin operation without alternation of the structure at any point of the refinement significantly reduced the calculated R factors. We thus report the final R factors for both with (-k, -h, -l) and without twin operation in Table 1.

Table 1
Crystallographic data collection and refinement statistics*

RNA binding and cleavage assay

The RNA cleavage activity assay was carried out in a volume of 20 μL containing 1 μM MmCas6b, 16 nM 5′-32P-labeled RNA, 200 mM NaCl, 20 mM Tris-HCl pH 7.5. The cleavage reaction was terminated by adding 98% formamide at various time points. The cleavage products were separated on a 14% polyacrylamide gel and visualized by phosphorimaging.

The electrophoretic mobility shift assays were carried out with a 13% native polyacrylamide gel loaded with binding reaction mixtures containing 0.25–1.0 μM MmCas6b, 0.5 μM (d37mer) or 1 μM (14mer) RNA oligo, 200 mM KCl, 5% Glycerol; 0.5 mM Dithiothreitol (DTT); 0.5 mM Ethylenediaminetetraacetic acid (EDTA) and 10 mM Tris-HCl pH 8.0. The unbound RNA and retarded species were visualized by staining the gel with SYBR Gold (Life Technology) for 30 minutes in a Tris Acetate-EDTA buffer.

Nitrocellulose filter-binding experiments were carried out to determine binding constant. A trace amount of (1 nM) 5′-32P-labeled RNA was incubated with increasing amount of protein in a 100 μL binding buffer containing 20 mM Tris-Cl pH 7.5, 100 mM NaCl, and 5% glycerol for 1 hour. The mixture was loaded into wells of MINIFOLD II Slot-blot system (Schleicher & Schuell) and passed through nitrocellulose and nylon filter membrane sequentially after applying vaccum. The filter was then washed with binding buffer and visualized by phosphorimaging. The fraction of bound was calculated from the intensity of the spot on the nitrocellulose membrane divided by the sum of the intensity of spots on the nitrocellulose and nylon membrane. The apparent dissociation constant was determined by fitting the binding isotherm to the Hill equation:

θ=[P]nKd+[P]n

where θ is the fraction of bound, n is the Hill coefficient, Kd is the binding constant and [P] is the concentration of MmCas6b.

Photo-cross-linking and high –resolution mass spectrometry

UV-induced protein – RNA cross-linking was performed essentially as described (Kramer et al., 2014). Briefly, 1 nmol of the Cas6b protein was mixed with 1 nmol of the repeat RNA and incubated on ice for 30 min for complex formation. The complexes were transferred to black polypropylene microplates (Greiner Bio-One) and UV irradiated at 254 nm for 10 min on ice at a distance of 1 cm from the UV lamp. The samples were precipitated with ethanol and the pellet was dissolved in 4 M Urea and 50 mM Tris-HCl pH 7.9 which was then diluted to 1 M with 50 mM Tris-HCl pH 7.9. The RNA was hydrolyzed using 1 μg Benzonase for 2 hours at 37°C in presence of 1 mM MgCl2. Following RNA digestion, the sample was digested with trypsin (Promega) with an enzyme to substrate ratio of 1:20 at 37°C overnight. To remove non cross-linked RNA fragments the sample was desalted using in-house prepared C18 (Dr. Maisch GmbH) columns and the cross-linked peptides were enriched using an in-house prepared TiO2 column (GL Sciences). The samples were then vacuum dried and dissolved in 12 μL 5% v/v ACN in water, 1% v/v FA in water for LC-MSMS analysis. 8 μL were injected onto a nano-LC system (Agilent 1100 series, Agilent Technologies) coupled with an LTQ-Orbitrap Velos instrument (Thermo Fisher Scientific). ESI-MS was performed in data-dependent mode using a TOP10 HCD method. The precursor ions as well as fragment ions were scanned in Orbitrap, and the resulting spectra were measured with high accuracy (< 5 ppm) both in the MS1 and MS2 level. Data analysis was performed as described previously (Kramer et al., 2014) and the high scoring cross-linked peptides were manually annotated for confirmation.

Highlights

  • Cas6 from Methanococcus maripaludis binds to two sites of the long CRISPR repeat
  • M. maripaludis Cas6 recognizes a two-base pair stem and a AAYAA loop
  • Cas6 supplies a tryrosine residue as a nucleobase mimic that bases with an adenine
  • The productive dual-binding by Cas6 competes with a non-productive RNA structure

Supplementary Material

1

Acknowledgments

This work was supported by National Institutes of Health grant R01 GM99604 to H.L. and the Deutsche Forschungsgemeinschaft (DFG FOR 1680) to L.R. and H.U.. X-ray diffraction data were collected from the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions for APS beamlines may be found at http://necat.chem.cornell.edu/ and http://www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the UnitedStates Department of Energy Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38.

Footnotes

AUTHOR CONTRIBUTION

Y.S. and H.L. designed and performed crystal structure determination. H.R. and L.R. cloned MmCas6b and performed mutagenesis analyses. K.S. and H.U. performed mass spectrometry analyses. Y.S. and H.L. wrote and all other authors edited the paper.

ACCESSION NUMBERS

The crystal structures of MmCas6b-RNA complexes were deposited in the PDB database under the accession number 4Z7K for the d31mer complex and 4Z7L for the 14mer complex, respectively.

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