
==== Front
Nucleic Acids Res
Nucleic Acids Res
nar
Nucleic Acids Research
0305-1048
1362-4962
Oxford University Press

39180400
10.1093/nar/gkae717
gkae717
AcademicSubjects/SCI00010
Structural Biology
Structural insights into RNA cleavage by a novel family of bacterial RNases
Wu Ruoxi Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

Ingle Shakti Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

Barnes Sarah A Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

Dahlin Heather R Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

Khamrui Susmita Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

Xiang Yufei Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

Shi Yi Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

https://orcid.org/0000-0002-8502-2430
Bechhofer David H Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

https://orcid.org/0000-0002-6738-2563
Lazarus Michael B Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

To whom correspondence should be addressed. Tel: +1 212 241 7770; Fax: +1 212 996 7214; Email: michael.lazarus@mssm.edu
Correspondence may also be addressed to David H. Bechhofer. Tel: +1 212 241 5628; Fax: +1 212 996 7214; Email: david.bechhofer@mssm.edu
23 9 2024
24 8 2024
24 8 2024
52 17 1070510716
6 8 2024
30 7 2024
29 2 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.
2024
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Abstract

Processing of RNA is a key regulatory mechanism for all living systems. Escherichia coli protein YicC belongs to the well-conserved YicC family and has been identified as a novel ribonuclease. Here, we report a 2.8-Å-resolution crystal structure of the E. coli YicC apo protein and a 3.2-Å-cryo-EM structure of YicC bound to an RNA substrate. The apo YicC forms a dimer of trimers with a large open channel. In the RNA-bound form, the top trimer of YicC rotates nearly 70° and closes the RNA substrate inside the cavity to form a clamshell-pearl conformation that resembles no other known RNases. The structural information combined with mass spectrometry and biochemical data identified cleavage on the upstream side of an RNA hairpin. Mutagenesis studies demonstrated that the previously uncharacterized domain, DUF1732, is critical in both RNA binding and catalysis. These studies shed light on the mechanism of the previously unexplored YicC RNase family.

Graphical Abstract

Graphical Abstract

National Institute of General Medical Sciences 10.13039/100000057 R01GM147211 R35GM124838 R35GM137905 National Institutes of Health 10.13039/100000002
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pmcIntroduction

Dozens of bacterial ribonucleases that are involved in the processing and turnover of all types of RNA have been discovered in recent years, with the surprising finding that the complement of RNases in different organisms can vary greatly. For example, for the model Gram-positive Bacillus subtilis and Gram-negative Escherichia coli bacteria, nine of the known B. subtilis RNases are also found in E. coli, whereas 12 other B. subtilis RNases are not found in E. coli (1). There are typically redundant functions for several RNases of a particular class (e.g. endonuclease, 3′ exonuclease), such that the deletion of an RNase-encoding gene may not show a strong phenotype.

Recently, our laboratory discovered a novel endoribonuclease in B. subtilis, YloC, in a strain that was deleted for genes encoding the four known 3′ exoribonucleases (2). YloC is a divalent cation-dependent hexameric endonuclease whose in vivo function is unknown. The identity of YloC as an endoribonuclease was a surprise, as one could not predict this from its sequence, which is unrelated to known ribonucleases. YloC belongs to the so-called YicC family, named after the E. coli homologue, with which it shares 30% identical residues. YloC and YicC catalyze the same endonuclease reaction in vitro (2).

YicC family members have been implicated in several areas of bacterial physiology, including stress response (3), sporulation (4), iron regulation (5) and DNA repair (6). For example, prior to the discovery of YloC, E. coli YicC was identified in a genetic screen for its role in the degradation of the small regulatory RNA RyhB (5). YicC-like proteins are widespread in bacterial species and have highly conserved N-terminal and C-terminal domains. The prototypical member of the family is E. coli YicC, a 33 kDa protein that consists of several domains, including an N-terminal domain, a central α-helical section, and a conserved C-terminal domain of unknown function ‘DUF1732,’ so-called because it is without a reliable annotation in the Pfam database (7). DUF1732 is in the top 20 of DUFs that are present in 500 or more species (8). While we were preparing this manuscript, a low resolution YicC crystal structure at 4.05 Å was published, and the structure confirmed that the protein forms a hexamer (9). However, how YicC family members bind and cleave RNA substrates remains unknown.

Here, we report a 2.8-Å-crystal structure of E. coli YicC, providing a detailed view of the apoprotein. We also report a cryo-EM structure of YicC bound to a 26-nucleotide (nt) RNA substrate at 3.2 Å resolution. We performed fluorescent anisotropy, mass spectrometry and mutagenesis studies to investigate RNA cleavage by YicC. Our structural and biochemical studies provide insight into RNA recognition and catalysis by YicC, shedding light on the ribonuclease activity of the YicC RNase family.

Materials and methods

Cloning, protein expression and purification

For expression of B. burgdorferi YicC protein, the 873-nt coding sequence was synthesized (Twist Biosciences) and amplified by PCR, followed by Gibson cloning into the modified pET47b vector used for the other YicC proteins. For apo YicC purification for crystallography, we purified the protein as previously described (2). Briefly, an overnight culture of the YicC expression strain was diluted 1:100 in 3 l of LB medium containing 1 mM MgSO4, and the culture was grown to OD600 of 0.5. YicC expression was induced by addition of IPTG to 400 μM, followed by further incubation with shaking for three hours. Cell were washed with 20 mM Tris, pH 8.0, 250 mM NaCl and cell pellets were stored at −80°C. Cells were resuspended in TBS (20 mM Tris, pH 8.0, 150 mM NaCl). PMSF was added to 1 mM and lysozyme was added to 100 μg/ml. The cell resuspension was sonicated (Qsonica) to lyse cells and centrifuged at 36 000 x g for 30 min. The supernatant was loaded onto Ni-NTA resin (0.5 ml/l culture, Qiagen) that was pre-equilibrated with TBS plus 25 mM imidazole. After 1 h incubation, the resin was washed with TBS plus 50 mM imidazole and eluted with TBS plus 250 mM imidazole. After overnight cleavage by SUMO-protease, the target protein was loaded onto a Superdex 200 10/300 Increase Column (GE) in TBS. For cryo-EM, the protein was purified the same way except after overnight cleavage by SUMO-protease, the target protein was loaded onto a Hitrap Q column (GE) and eluted with a linear NaCl gradient, followed by size-exclusion chromatography (Superdex 200, GE) in buffer D (20 mM HEPES, pH 7.5, 150 mM NaCl and 5 mM TCEP). Peak fractions were pooled, concentrated, flash frozen in liquid nitrogen and stored at −80°C. To assemble the RNA-YicC complex, the RNA was incubated with YicC protein at a molar ratio of 1.5:1 at 4°C for 40 min. The complex was loaded onto a Superdex 200 column equilibrated with buffer D for further purification.

Purification of YicC protein for RNase and anisotropy assays

Cells were grown as described above. Frozen pellets from 100 ml of culture were resuspended in 15 ml ice cold TBS containing 250 mM NaCl. PMSF was added to 1 mM and lysozyme was added to 100 μg/ml. The cell suspension was sonicated and lysed cells were spun down at 36 000 × g for 30 min. The supernatant was added to 0.25 ml of Ni-NTA and 250 mM imidazole was added for a final concentration of 25 mM imidazole. The lysate and resin were rotated for 1 h at 4 °C. The resin was washed with 20 ml of 50 mM imidazole in TBS/10% glycerol, and protein was eluted with 250 mM imidazole in TBS/10% glycerol. Four buffer exchanges with TBS/10% glycerol were performed in an Amicon-ultra 4 concentrator, to a final volume of less than 250 μl. Protein concentration was determined using the Bradford reagent (BioRad), and protein was stored at −80°C. Protein purity was examined by PAGE. YicC proteins were judged to be >90% pure. For anisotropy experiments, the same protocol was scaled up for 2 l cultures.

Crystallography

After screening crystallization conditions, crystals were obtained and optimized by hanging drop vapor diffusion, using a reservoir containing 0.1 M Tris pH 8.5, 0.3 M lithium sulfate, and 25% PEG 3350. Data was collected at the NSLSII beamline AMX, and was processed using autoproc (26). The data was anisotropic, so we used the autoproc anisotropic processing, which diffracted to 2.8 Å in the best direction. The additional resolution from the anisotropic processing was critical in identifying side chains. We then solved the structure by molecular replacement, using the AlphaFold structure of YicC as a search model (10,27). The models were subsequently refined using Phenix (28) with rigid body refinement and multiple rounds of simulated annealing, minimization, atomic displacement parameter (ADP or B-factor) refinement and TLS refinement (determined using the TLSMD server) (29,30), with interspersed manual adjustments using Coot (31). All structural figures were made with Pymol (32). except for the cryo-EM electron density figure which was made with Chimera (33).

Electron microscopy

YicC-RNA complex sample at a concentration of 5 mg/ml was supplemented with 0.05% dodecyl maltoside detergent immediately before plunge-freezing, to enable even distribution of particles on the grid. 3 μl of sample was applied to glow-discharged Quantifoil holey carbon grids (Cu, R1.2/1.3, 300 mesh). The grids were blotted for 2 s and plunged into liquid ethane with a Vitrobot plunger (4°C and 90% humidity). Cryo-EM data were collected with a Titan Krios microscope (FEI) operated at 300 kV and images were collected at a nominal magnification of 81 000 corresponding to a pixel size of 1.07 Å with a defocus range of −0.5 to −2 μm. The images were recorded on a K3 electron direct detector in super-resolution mode at the end of a GIF-Quantum energy filter operated with a slit width of 15 eV. For data collection with a K3 detector, a dose rate of 15 electrons per pixel per second and an exposure time of 3.82 s were used, generating 70 video frames with a total dose of 60 electrons per Å2. The statistics for the cryo-EM data are listed in Supplementary Table S3.

Image processing

A total of 5347 dose-fractionated videos of YicC-RNA complex was collected. The processing was done within cryoSPARC (34,35). Motion correction was done by cryoSPARC’s Patch motion correction with an output F-crop factor of one-half. CTF estimation for each micrograph was calculated with Patch CTF estimation. Eight million particles were auto-picked and extracted from micrographs. Ab initio models were generated as initial references for subsequent 3D classifications. Then the particles were sorted by multiple rounds of two-dimensional (2D) classification and three-dimensional (3D) classification to exclude bad particles; all classes containing YicC-RNA density were combined (342 211 particles) and used for NU-refinement, resulting in a final map at 3.2 Å resolution.

Cryo-EM refinement

Using our crystal structure, we fit a model using Phenix dock_in_map and refined the structure with iterative manual adjustments in Coot and in Phenix real_space_refine, including B factor refinement and rotamer correction, using RNA restraints generated by Double Helix (36).

RNase assay

YicC enzyme assays were performed in a 50 μl volume of RNase assay buffer, which was 5 mM MgCl2, 50 mM Tris pH 8.0, 7 mM NaCl, 100 mM KCl, and 400 μg/ml BSA. Unlabeled (20 pmoles) and IR-fluorescent-labeled RNA (10 pmoles) were used, for a final concentration of 600 nM. For fluorescent assay of 26-mer cleavage, unlabeled and labeled RNAs were DHB2051 and DHB2150 (Supplementary Table S1). For the 36-mer, the unlabeled and labeled RNAs were DHB1866 and DH1879 (Supplementary Table S1). YicC protein, containing the N-terminal Sumo tag, was added to a final concentration of 20 nM, giving a 1:30 protein:RNA ratio, except for the 36-mer, for which YicC protein was added to a final concentration of 100 nM, giving a 1:6 protein:RNA ratio. For the B. burgdorferi assay, YicC protein was at a concentration of 160 nM, giving a 1:3.75 protein:RNA ratio. At time points after addition of YicC, 10 μl of the reaction were removed into an equal volume Gel Loading Buffer II (Invitrogen) on ice to stop the reaction. Half of each sample (10 μl) was separated on a 20% denaturing polyacrylamide gel (Sequagel; National Diagnostics). Reaction products were visualized on a LI-COR Odyssey CLx imaging system and analyzed using LI-COR Image Studio software.

Mass spectrometry

After endonuclease digestion, the RNA was isolated by phenol–chloroform extraction before resuspension in 30% LC/MS grade methanol at approximately 125 μM concentration. After 10× dilution, 200 μl RNA was loaded by a syringe pump for direct infusion. RNA was ionized by a heated electrospray ionization (HESI) probe and was analyzed with an Exploris 480 Orbitrap™ mass spectrometer (ThermoFisher). The flow rate was set at 10 μl/min. The instrument was operated in the negative ionization mode at 3.0 kV for both MS and MS/MS acquisitions. The MS scan range was set at 500–6000 m/z and the spectra were acquired at 240 000 resolution (at m/z = 200). Specific RNA precursor ions were selected for tandem MS (MS (2)) acquisition with an isolation window of 3 Th and maximum injection time of 100–800 ms. Different high-energy collisional dissociation (HCD) energies of 20%, 25% and 30% were evaluated to facilitate better fragmentation. After acquisition, the spectra were analyzed by a script from Ariadne (https://ariadne.riken.jp/index.html) and manually validated.

Fluorescence anisotropy

Cy3-labeled RNA (DHB1865; Supplementary Table S2) was used at a fixed final concentration of 50 nM. All YicC proteins, which carried the N-terminal Sumo tag, were diluted twofold serially in TBS/10% glycerol, pH 8.0, from 175 μM final concentration across 11 concentrations and a 12th at no protein, except for E281A protein, which was diluted starting at 9 μM. After mixing protein in triplicate with RNA on ice, the fluorescent polarization was read on a Victor NIVO plate reader using excitation at 545 nM, emission at 635 nM, and a 595 nM dichroic mirror. The Kd values in Supplementary Table S5 were calculated using Graphpad Prism and a log IC50 fit, from the data shown in Figure 5.

Results

Crystal structure of apo YicC

In order to gain insight onto the function and mechanism of this new family of endoribonucleases, we undertook structural studies of the B. subtilis YloC protein and the E. coli YicC protein. We were unable to obtain crystals of the B. subtilis protein YloC; we therefore focused on YicC. After purifying the protein (Figure 1A), which we previously reported forms a hexamer in solution (2), we were able to obtain crystals of YicC that diffracted to 2.8 Å anisotropically. We then solved the structure using the AlphaFold (10) predicted model of YicC as a search model and refined the structure to good Rfree and stereochemical statistics (Supplementary Table S1). The monomer of YicC forms an extended hook that consists of three regions: a scaffold region, a hinge region and a cap region (Figure 1B and C). The scaffold region comprises an N-terminal β-strand (residues 1–70), a central α-helical stalk (126–213) and the DUF1732 (DUF) domain (214–287), with the N-terminal β-strand and the DUF domain flanking the central α-helical stalk. In our crystal structure, YicC indeed forms a hexamer, consisting of a dimer of trimers (Figure 1D). One trimer lies on top of the other trimer with about a 70-degree rotation to form an open and twisted conformation (Supplementary Figure S1). The overall structure stretches 110 Å in length, with the ends of the two trimers 75 Å apart (Figure 1D). Six α-helices from different monomers are oligomerized into a hexagonal cap (82–112) at the end of the structure. The cap domain facilitates hexamerization through a series of intermonomer salt bridges (Supplementary Figure S2). The scaffold region and the cap region are connected by the hinge region that is composed of flexible loops (71–81 and 113–125), as evident in the higher B-factors for this region. The scaffold region includes both the central α-helical stalks and DUF1732 domains, which are arranged in ternary structure to form a pincer-like assembly with a channel large enough to accommodate an RNA molecule.

Figure 1. Crystal structure of apo YicC. (A) Purification of YicC, with chromatogram from a Superdex 200 Increase column and Coomassie gel of peak fractions (35 and 40 kDa molecular weight markers indicated). (B) Crystal structure of YicC monomer. (C) Domain architecture of YicC protein and its homologs. The different domains and regions are colored; the color scheme is used for all panels in this figure. (D) Crystal structure of YicC from three different angles in cartoon representation. The opening was measured from B chain L196/CA to E chain Q195/CA.

Within each trimer, two YicC monomers from either side are arranged around the central monomer to form a crescent-shaped scaffold. The DUF domain consists of three parallel α-helices, and the central DUF copy makes extensive hydrogen bonds with residues from two side DUF copies, as shown in Supplementary Figure S3. This domain is largely responsible for the trimerization interface, although the cap and central stalk also participate in forming trimerization contacts. We also noticed several density areas that formed along a series of arginine residues in the cavity. We assigned these to sulfates from the crystallization condition but hypothesize that they are mimicking the phosphates from the RNA backbone that would interact with the multiple basic residues in the cavity (Supplementary Figure S4).

Cryo-electron microscopy structure of YicC bound to an RNA substrate

To study RNA recognition and catalysis by YicC, we co-purified YicC and a 26-nt RNA substrate by gel filtration, in the absence of Mg2+ to prevent cleavage (DHB2051, Supplementary Table S2). This particular RNA substrate was chosen because preliminary work with the B. subtilis YloC protein showed that DHB2051 bound more strongly than several other RNA oligonucleotides that were tested. We collected cryo-EM data and obtained a reconstruction of YicC in complex with substrate RNA that was refined to 3.2 Å overall (Supplementary Figures S5–S7, Supplementary Table S3), including well-defined density for RNA (Figure 2A). A model was generated from our crystal structure of YicC and by building the RNA manually. Surprisingly, the YicC hexamer undergoes a significant conformational change when it binds to RNA (Supplementary Figure S8 and Supplementary Movies S1 and S2), pivoting at the cap region. While in the apo structure, one trimer lies on top of the other trimer in an open and twisted position, in the complex, the top trimer rotates nearly 70 degrees towards the bottom trimer to form a barrel. This conformation resembles a clamshell that encloses the substrate RNA inside like a pearl (Figure 2B). The RNA closed complex is a pseudo-dimer, with one trimer recessed and the other one extending outward. This unique asymmetry likely allows the hexameric complex to present different sets of residues to each side of the hairpin.

Figure 2. Cryo-EM structure of YicC bound to an RNA substrate. (A) Electron density of the structure, colored by chain. (B) Overall model of YicC-RNA complex. The structure is shown in cartoon representation, colored by chain. The RNA is shown in stick format, in the protein cavity. (C) surface view of YicC-RNA complex. One trimer has been removed for clarity, and the structure is colored by domain, as in Figure 1. (D) Schematic of RNA structure, with basepairing indicated. The loop that is not observed in the structure is indicated by dotted lines. (E) Structure of RNA shown in stick form, with bases numbered and the missing loop indicated. The central cavity was measured by I212B/CA to Y35B/CA, which is 53.7Å

Substrate RNA binds in the central cavity

The RNA-bound YicC structure possesses a substrate cavity that is organized by three domains: the N-terminal β-sheet, the DUF region and the central α-helical stalk. Six copies of each domain coordinate together to wrap around the central cavity, creating a 50-Å-long × 30-Å-wide barrel, in which the RNA substrate resides (Figure 2C). The resolution was sufficient to identify the RNA bases and protein sidechain interactions with the RNA (Supplementary Figures S9 and S10). The 26-nt RNA adopts a hairpin conformation and predominately occupies the positively charged region of the cavity, with the 5′-proximal hairpin pointing to the closed tip and the 3′ single strand extending towards the cap. RNA nt 2–16 form the hairpin region, with nt 7–11 forming the loop (Figure 2D). Because the density of nucleotides in the loop region is not well defined due to its flexibility, we were unable to build that part of the RNA (Figure 2E). The 3′ tail is also flexible and the density for nt 23–26 was not sufficient for building this portion of the RNA.

Inside the barrel, the N-terminal domain and DUF domain form a positively charged patch that can bind RNA through electrostatic interactions (Figure 3A). As a pseudo-dimer, YicC residues interact with RNA asymmetrically inside the cavity (Figure 3B and C). While we observe several amino acids (R30/F, R280/E, R251/A, T255/A, S258/A and R211/A) interacting with 5′-proximal RNA nucleotides, even more residues (R30/D, R280/B, R30/B, R240/B, R30/A, R67/F, N254/B, N254/A and S258/A) are arranged along the downstream side of the hairpin and the 3′ single-stranded sequence, interacting with the RNA backbone through hydrogen bonding and electrostatic interactions. Many of the arginine residues that we observed in the apo structure interacting with sulfate ions, primarily R30 and R280 from multiple chains (Supplementary Figure S4), are observed here to interact with the RNA backbone. Much of the RNA binding interface comes from the DUF domain, which wraps around the hairpin region (Figure 3D).

Figure 3. Structural determinants of RNA binding. (A) Overall structure of YicC-RNA complex shown with electrostatic potential, with positive charge shown in blue and negative charge in red. (B) Closeup of protein-RNA interactions, with side chains indicated and potential hydrogen bonds and ionic interactions shown. Three different segments of the RNA, labeled as I, II and III are shown. (C) Schematic of amino acid–RNA interactions, with the residue number and chain indicated after the slash, colored by domain. (D) Axial view of the DUF–RNA interactions, with the RNA shown in spherical representation.

RNA is cleaved at two sites by different YicC family members

To monitor YicC cleavage of RNA, we used an IR-fluorescent labeled version of the RNA that was in the cryo-EM structure (DHB2150, Supplementary Table S2). The reaction mixture contained also unlabeled 26-nt RNA to increase the RNA concentration to 600 nM. With a 5′ label, we were able to follow cleavage of the RNA oligonucleotide (oligo) over a short time course (Figure 4A). We observed two smaller bands that both increased with time. The smaller cleavage product B is the major product, but the larger product C seen on the gel also increases over time. A cleavage assay with longer time points, up to 64 minutes, indicated that product C is an end product and is not cleaved further (Supplementary Figure S11). In this assay we can only observe fragments that contain the 5′ labeled end, but cleavage at two sites suggests that there could be as many as five fragments in total generated by the enzyme (Figure 4B, fragments B–F).

Figure 4. RNA cleavage activity of YicC and site identification. (A) Fluorescent cleavage gel assay. Total RNA concentration was 600 nM; YicC protein concentration was as indicated above each set of reactions. Reactions were incubated at 37°C and aliquots were removed at times indicated above each lane. Control lane (–), no YicC protein added. Migration of full-length RNA oligo (A) and cleavage products (B and C) indicated at left. (B) Schematic of cleavage fragments resulting from two cleavages in DHB2051. Predicted RNA fragments are labeled A-F. Asterisk indicates the site of the fluorescent dye modification for gel experiments. For the mass spectrometry experiments they are unmodified hydroxyl 5′ ends. (C) Time course of cleavage activity on 26-mer RNA for E. coli YicC, B. subtilis YloC, and B. burgdorferi (Bb) YicC. RNA concentration was 600 nM; YicC protein concentration indicated above each set of reactions. Aliquots were removed at times (min) indicated above each lane. Control lane (–), no protein added. Migration of full-length RNA oligo (A) and E. coli YicC cleavage products (B and C) indicated at left. (D) High-resolution mass spectrometry analysis of cleavage of DHB2051. The reaction was purified and the sample (∼10 μM) was directly infused to an orbitrap mass spectrometer and analyzed under the negative ionization mode. Upper panel shows the full MS spectrum of the RNA analytes at 400–1150 m/z. Zoom-in of three dominant RNA species including salt adducts (590–680 m/z) is shown in the lower panel. Their precise m/z measurement, respective molecular compositions and mass accuracies in parts per million (ppm) are denoted. (E) Schematic showing cleavage sites of RNA hairpin by YicC, marked by red arrows. We hypothesize that the site 2 cleavage occurs on an alternative hairpin structure (see Discussion). (F) Proposed catalytic site, shown as zoomed-in view. The cluster of three glutamates are shown along with presumed water molecules shown as green spheres. The nucleotides between which site 1 cleavage occurs are shown in yellow.

We reported previously that YloC of B. subtilis and YicC of E. coli cleave a particular RNA substrate at the same positions (2). The homology between the YicC and YloC protein sequences is relatively high: 30.2% identity and 52.9% similarity. We noticed that the homology between the YicC-family protein from Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease, and E. coli YicC was less: 25.6% identity and 48.2% similarity. It was of interest to determine whether the B. burgdorferi YicC family protein also had similar ribonuclease activity. We therefore analyzed the cleavage patterns for E.coli YicC, B. subtilis YloC, and B. burgdorferi YicC (Figure 4C). (The B. burgdorferi YloC protein was used at a higher concentration as it showed no cleavage activity at 20 nM concentration; Supplementary Figure S12.) Although the B. burgdorferi YicC pattern showed an additional band migrating at the position of fragment C, the overall pattern was similar to that of E. coli YicC and B. subtilis YloC. This indicated a conserved cleavage specificity among diverse YicC family members.

Next, to investigate the dependence of YicC cleavage on RNA size and sequence, we employed an IR-fluorescent labeled 36-nt RNA that was used previously to detect ribonuclease activity in B. subtilis extracts (2). The 36-nt RNA (DHB1879, Supplementary Table S2) had a different sequence from the 26-nt substrate used in structural studies. We assayed cleavage of this RNA, side-by-side with cleavage of the 26-nt RNA (Supplementary Figure S13). YicC was less active on the 36-nt RNA, and five times as much protein was needed to see significant accumulation of cleavage products. The cleavage pattern of the 36-mer was more complex than for the 26-mer, but overall both substrates were cleaved proximal to the 5′ end and some distance downstream.

YicC cleaves across the hairpin with a glutamate triad in the active site

In order to determine the exact size and composition of the fragments generated by YicC cleavage, we turned to mass spectrometry to monitor the unlabeled cleavage reaction, with the same 26-mer RNA substrate that was used in the cryo-EM structure. After the cleavage reaction, RNA products were purified by phenol-chloroform extraction and ethanol precipitation. To assess the precipitation of small RNA fragments, we performed a reaction in parallel that contained the unlabeled 26-mer RNA and included a trace amount of 5′-end labeled 26-mer RNA. As could be expected, we found that the 5′-terminal 3-nt fragment B could not be precipitated (Supplementary Figure S14). The purified reaction products were analyzed by mass spectrometry, and we were able to observe three major products: the 14-nt fragment (from 1–14, fragment C), the 11-nt fragment (from 4–14, fragment D), and the 12-nt fragment (from 15–26, fragment E). The mass spectra had the precise masses and fragmentation patterns corresponding to the RNA sequence and predicted cleavage sites (Figure 4D and Supplementary Figure S15). The mass spectrometry data in combination with the gel fluorescent assay and the structural information reveals that YicC cleaves between C3 and A4 and between U14 and G15 (Figure 4E).

Initially, fragment F was not detected by ESI/mass spectrometry (MS). We hypothesized that this was either due to poor ionization under the negative ionization mode, low abundance, or immediate sequential cleavage at sites 1 and 2. To investigate these possibilities, we performed additional experiments. First, using a synthetic fragment F (nt 4–26), we confirmed that it was well-ionized on our Orbitrap MS instrument (Methods, Supplementary Figure S16A). Next, we incubated fragment F with YicC and readily detected fragments D (nt 4–14) and E (nt 15–26) (Supplementary Figure S16B), demonstrating that fragment F can be cleaved at site 2. Finally, we cleaved the full-length fragment (nt 1–26) for 12 min at room temperature to slow down the reaction and analyzed the early reaction products. As seen in Supplementary Figure S16C, fragment F was detectable at early time points, while other fragments were much less abundant. These results demonstrate that cleavage preferentially occurs at site 1, and the resulting 3′ fragment F can subsequently be cleaved at site 2.

Knowing the exact location of site 1 YicC cleavage, we examined our structure to identify potential catalytic residues. In the cryo-EM structure of the YicC-RNA complex, a cluster of three glutamates E216/A, E217/A and E281/F were observed in close proximity (<5 Å) to bases C3 and A4 (Figure 4F). Close to the glutamate triad, we observed two spheres of electron density (Supplementary Figures S17A and S17B). We assigned these to water molecules but believe they could be the sites of magnesium ions in a catalytic complex, since we know the ribonuclease activity is divalent-cation dependent (2). On the other side of the RNA hairpin there were additional glutamates and aspartates, but they were not sufficiently close to the RNA, in the conformation we observe in the cryo-EM structure, to suggest a catalytic site (Supplementary Figure S17C).

Mutagenesis and biophysical assay identify YicC residues involved in catalysis and binding

To test the involvement of specific YicC residues, targeted mutagenesis was performed; the targeted residue was changed in each case to alanine. Residues chosen for mutagenesis were those hypothesized to be involved in catalysis or RNA binding, or highly conserved (2) in YicC-family members. Fifteen mutant proteins were expressed and purified, and RNase activity was assessed using the gel assay, as in Figure 3A, with aliquots removed after 1 min and 16 min. The mutants expressed well and their concentrations were normalized before all assays (Supplementary Figure S18). The data in Figure 5A show that wild-type YicC cleaves a significant amount of the RNA oligo after 1 min and almost 100% of the substrate at 16 min. A similar pattern was seen in four of the mutants: E148, Q247, E277 and E287—indicating that these residues are not involved in RNA binding or catalysis and are not essential for protein folding. Ten of the mutants showed no cleavage activity in this assay: four of these were changes of glutamate residues (E216, E217, E252, E281), which we hypothesized were involved in catalysis, and three of these were changes of arginine residues (R30, R251, R280), which we hypothesized were involved in RNA binding (Supplementary Figure S4). The lack of activity for these mutants supported these hypotheses. The other three residues—M5, N28 and F244—were targeted based on their conservation across species and may be involved in helping to position the catalytic and binding residues. As shown in Supplementary Figure S19, the mutations we explored covered the RNA binding surface extensively, since each mutation affects all six monomers. Interestingly, the R211 mutant gave reduced RNase activity and appeared to yield more fragment C relative to fragment B. To confirm this, a time course was performed, comparing wild-type YicC to the R211 mutant (Supplementary Figure S20). The result showed that, indeed, the ratio of cleavage products for the R211 mutant was inverted with respect to wild type. Although R211 is >10 Å away from the cleavage site, it is possible that the binding of the RNA and presentation to the catalytic site is altered by the R211 mutation.

Figure 5. Analysis of catalysis and substrate binding. (A) Mutagenesis and activity measurements. Alanine mutants of the indicated residues were generated. DHB2051 RNA concentration was 600 nM; YicC protein concentration was 20 nM. Reactions were incubated at 37 °C and aliquots were removed at 1 min and 16 min. The YicC residue that was mutated to alanine is indicated for each mutant protein. Control lane (–), no YicC protein added. (B) Analysis of YicC mutant binding to RNA by fluorescence anisotropy. Binding constants were measured using a fixed concentration of 5′-Cy3 labeled RNA 36-mer and varied concentrations of wild-type or mutant YicC protein. Fluorescent polarization was measured on a multimode plate reader. The binding constants are shown in Supplementary Table S4. n = 3 for each data point, shown with error bars for standard deviation.

To demonstrate whether YicC mutant proteins that showed no RNase activity were defective for catalysis or for RNA binding, we performed fluorescence anisotropy experiments to measure the Kd of RNA-protein binding for three glutamate and three arginine mutants. The RNA substrate in this case was a 5′-Cy3 end-labeled 36-mer RNA. This RNA substrate gave a 5 μM Kd value for the wild-type protein. Remarkably, all three arginine mutants showed greatly impaired binding, while the three glutamate mutants showed slightly better or even much better binding (Figure 5B and Supplementary Table S5). We hypothesized that the glutamate mutants bind to the RNA better due to less charge-charge repulsion in the absence of cation; however, when we tried the anisotropy experiment in the presence of 10 mM CaCl2 (a cation that does not enable YicC catalysis) there was no change in the binding of either the WT or mutant protein (Supplementary Figure S21). We cannot exclude the possibility that YicC is not binding CaCl2, which is why it does not enable catalysis. In any event, the loss of RNase activity for the glutamate mutants is due to an effect on catalysis, while the loss of RNase activity for the arginine mutants is due to deficient RNA binding. The R211A mutant binds less well than wild-type but still has catalytic activity. The mutational results are summarized in Supplementary Table S6. We conclude that the cluster of glutamate residues, including E216, E217, E252, and E281, is likely involved in catalysis by coordination of Mg2+ ions (where we observed water molecules in our cryo-EM structure), while the cluster of arginine residues presenting on the downstream end of the hairpin, including R30, R251 and R280, are likely involved in RNA binding by electrostatic interactions.

Discussion

That YicC specifies endoribonuclease activity could not be predicted from its primary sequence, as there is little homology to any of the known ribonucleases. Solution of the YicC apoprotein and YicC-RNA complex revealed a novel RNA binding mechanism. The apoprotein resembles a hinged container, similar to a clamshell, with an interior cavity large enough to contain an RNA sequence—in our case, 26 nt long. The structure is formed from six copies of the protein that are held together by the cap domain, which is encoded in the middle of the protein. A detailed analysis of the structure showed that the DUF1732 domain, whose function was previously unknown, participates in trimerization and contains many of the residues involved in RNA binding and catalysis. In the RNA–YicC complex, the clamshell has closed onto the RNA, with a set of arginine residues binding the RNA and exposing the hairpin to a set of glutamate residues that likely coordinate Mg2+ ions. This is a novel mechanism for RNA binding. Other endoribonucleases, such as RNase E (Supplementary Figure S22A) and RNase III (Supplementary Figure S22B), typically bind on the outside of an RNA structure (11,12). In the case of RNase E, RNA extends outside of the active site in each monomer of the tetrameric structure (Supplementary Figure S22A) (13). Another RNase that binds RNA entirely inside a cavity is the 3′ exonuclease PNPase, which forms a trimer as the active enzyme. However, in the case of PNPase, the RNA binds to a symmetric multimer, without much conformational change occurring upon binding (14,15). YicC, on the other hand, binds a single RNA asymmetrically and undergoes substantial conformational change between apo and bound states. It is noteworthy that Nsp15, the endoribonuclease from SARS-Cov2, which is the only other known hexameric endoribonuclease, binds six copies of RNA (16), further highlighting the unique nature of YicC. YicC, therefore, represents a new paradigm for ribonuclease binding of an RNA. Remarkably, the active site formed for cleavage consists of residues from two different trimers from the apo protein that come together to form a new active site. Glu 216 and Glu 281 from the cleavage site are >50 Å apart in the apo structure. Lastly, we note that the previously undefined DUF domain contains most of the key residues for binding and catalysis, showing that it a truly novel RNase domain.

Based on our combined structural, mass spectrometry, biophysical and biochemical data, we can propose a catalytic mechanism. The glutamates that are located near the 5′ cleavage site are essential for catalysis, likely through coordinating magnesium ions. This is reminiscent of the two metal-ion mechanism of RNase H (17,18). RNase III also cleaves hairpin RNAs using a two Mg2+ catalytic site (19,20). An SN2 nucleophilic reaction at the RNA phosphate backbone has been proposed to be the common catalytic mechanism in several bacterial endoribonucleases families, including RNase E (21), RNase III (19) and RNase H (22), in which a nucleophilic water attacks the phosphate backbone. Therefore, it is appealing to speculate that YicC uses a similar mechanism. The water is activated by one magnesium ion for nucleophilic attack, and the two magnesium ions, bound by the carboxylic acid sidechains, act to stabilize the negative charge of the transition state (23). Many of these RNases, including the DEDD superfamily of exoribonucleases, employ four acidic residues to promote catalysis (24). In the YicC family, there are three glutamates that are aligned at the 5′ cleavage site (Figure 4F), but YicC has no sequence relation to the other families, and so is perhaps an example of convergent evolution. We note, however, that we have not yet established that YicC operates by a similar mechanism. Future work will validate the position of the cations and determine the catalytic mechanism.

Initially, based on the observed products from 5′-labeled RNA substrates, we believed that cleavage could occur at either of two sites on different sides of the RNA hairpin, and that these cleavages were independent of each other. However, further mass spectrometry experiments showed that cleavage at site 1 is both highly preferred and on the pathway for cleavage at the second site. This suggests that the minimal cleavage event is a single cleavage at the upstream side of an RNA hairpin. We hypothesize that after this cleavage, the RNA can adopt a new secondary structure that enables a second cleavage event. This also predicts that some substrates will only be cleaved a single time. Intriguingly, prediction of the secondary structure of fragment F (Supplementary Figure S23) shows a hairpin exactly where the cleavage is supposed to happen, mimicking the structure and cleavage at site 1. Future structural work will be needed to confirm this second hairpin hypothesis. We also do not know if YicC has any sequence specificity or merely requires a secondary structure of a certain length. Our results are consistent with the secondary structure hypothesis being most relevant, since there are no contacts to the RNA bases in our structure, except for the initial guanine and A19.

The result In Supplementary Figure S13, comparing cleavage of a 26-mer and a 36-mer, suggests that even for an RNA substrate with a different sequence and likely different structure, YicC still cleaves at two sites, one 5′ proximal and one more distal. The trio of small, 5′ proximal products may indicate that the 36-mer substrate is not bound by the enzyme as well as the 26-mer, and therefore there is some heterogeneity in the actual cleavage site. In any event, these data allow us to hypothesize that the in vivo targets of YicC may be the 5′-proximal structure of RNAs that need to be degraded, and YicC serves to remove a protective 5′ end. Future work will be necessary to explore more sequences to understand the relationship between sequence and cleavage. However, we hypothesize that a hairpin structure is necessary for cleavage by YicC in the upstream side of the hairpin.

As we did here, Huang et al. (9) also identified several arginine residues that contribute to RNA binding, including R30 and R280. Using a set of related RNA sequences as substrates, these authors reported that YicC recognizes primarily a GUG sequence. Our RNA substrates did not contain this trinucleotide sequence but were nevertheless cleaved precisely at two sites by YicC. We note that our ribonuclease time-course assays, which contained a 1:30 protein:RNA ratio, were quite different from those of Huang et al., who examined only a single 30 min time point and used a 1:1 protein:RNA ratio.

Our results with YicC-family proteins from three organisms showed a strong conservation of endonuclease specificity (Figure 4C). The cleavage pattern from E. coli YicC and B. subtilis YloC is identical, despite these two organisms being at opposite ends of a bacterial phylogenetic tree (25). The homology between these two proteins is relatively high. To demonstrate enzyme activity in a YicC-family protein from a different source, we cloned and expressed the B. burgdorferi YicC, which has less homology to E. coli YicC. In a phylogeny of bacterial ribonucleases, B. burgdorferi sits about midway between E. coli and B. subtilis and has only half as many ribonucleases as the other two organisms (25). The B. burgdorferi YicC protein cleaved the 26-mer substrate RNA similarly, on either side of its hairpin structure, although yielding slightly different sized fragments from those generated by the E. coli and B. subtilis enzymes (Figure 4C). The results with three YicC-family proteins from different locations on the bacterial phylogenetic tree suggest that YicC-family proteins from across the bacterial world will have endoribonuclease activity. Also, the two fluorescently labeled RNAs that were assayed here (Figure S6) showed a similar cleavage pattern, despite differences in RNA sequence and, most likely, structure. The capacity to accommodate and cleave different RNA molecules in a similar way may be a consequence of the clamshell-like ribonuclease mechanism.

While the function of YicC remains unknown, the conservation of the YicC family across bacteria and, as we show here, the conserved biochemical nature of family members’ endonuclease activity, suggests an important function for this enzyme. Efforts are underway to identify native substrates of this novel family of ribonucleases.

Supplementary Material

gkae717_Supplemental_Files

Acknowledgements

This work was supported by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) grants R35GM137905 (Y.S.), R01GM147211 (D.H.B.), and R35GM124838 (M.B.L). We thank Olga Rechkoblit for helpful suggestions on the structures. This research used beamline AMX of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. The Center for BioMolecular Structure (CBMS) is primarily supported by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) through a Center Core P30 Grant (P30GM133893), and by the DOE Office of Biological and Environmental Research (KP1607011). We used the Krios microscope at the Laboratory for BioMolecular Structure (LBMS), which is supported by the DOE Office of Biological and Environmental Research (KP1607011). Some molecular graphics and analyses were performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. This work was supported in part through the computational and data resources and staff expertise provided by the Scientific Computing and Data group at the Icahn School of Medicine at Mount Sinai and supported by the Clinical and Translational Science Awards (CTSA) grant UL1TR004419 from the National Center for Advancing Translational Sciences. Research reported in this publication was also supported by the Office of Research Infrastructure of the National Institutes of Health under award numbers S10OD026880 and S10OD030463. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Data availability

The data underlying this article are available in The Protein Data Bank at http://www.rcsb.org and can be accessed with deposition numbers 8VER and 8VES. The data underlying this article are available in MassIVE at https://massive.ucsd.edu, and can be accessed with accession number accession number MSV000095515.

Supplementary data

Supplementary Data are available at NAR Online.

Funding

National Institute of General Medical Sciences [R01GM147211, R35GM124838, R35GM137905]. Funding for open access charge: National Institutes of Health.

Conflict of interest statement. None declared.
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