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. 2006 Aug 11;361(2):243-56.
doi: 10.1016/j.jmb.2006.06.021. Epub 2006 Jun 27.

RNA recognition and cleavage by the SARS coronavirus endoribonuclease

Kanchan Bhardwaj  1 , Jingchuan Sun, Andreas Holzenburg, Linda A Guarino, C Cheng Kao
Affiliations

RNA recognition and cleavage by the SARS coronavirus endoribonuclease

Kanchan Bhardwaj et al. J Mol Biol. .

Abstract

The emerging disease SARS is caused by a novel coronavirus that encodes several unusual RNA-processing enzymes, including non-structural protein 15 (Nsp15), a hexameric endoribonuclease that preferentially cleaves at uridine residues. How Nsp15 recognizes and cleaves RNA is not well understood and is the subject of this study. Based on the analysis of RNA products separated by denaturing gel electrophoresis, Nsp15 has been reported to cleave both 5' and 3' of the uridine. We used several RNAs, including some with nucleotide analogs, and mass spectrometry to determine that Nsp15 cleaves only 3' of the recognition uridylate, with some cleavage 3' of cytidylate. A highly conserved RNA structure in the 3' non-translated region of the SARS virus was cleaved preferentially at one of the unpaired uridylate bases, demonstrating that both RNA structure and base-pairing can affect cleavage by Nsp15. Several modified RNAs that are not cleaved by Nsp15 can bind Nsp15 as competitive inhibitors. The RNA binding affinity of Nsp15 increased with the content of uridylate in substrate RNA and the co-factor Mn(2+). The hexameric form of Nsp15 was found to bind RNA in solution. A two-dimensional crystal of Nsp15 in complex with RNA showed that at least two RNA molecules could be bound per hexamer. Furthermore, an 8.3 A structure of Nsp15 was developed using cyroelectron microscopy, allowing us to generate a model of the Nsp15-RNA complex.

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Figures

Figure 1
Figure 1
RNA cleavage by the SARS-CoV Nsp15. (a) The image from a denaturing 20% polyacrylamide gel shows radiolabeled R16.4 treated over time with Nsp15 or with the inactive mutant H234A. At the time-point designated above the appropriate lane, an aliquot of the reaction was removed and stored in a sample dye containing urea until the entire set had been collected. The sequence of RNA R16.4 is shown above the gel image. The only uridylate, at position 13, is in a larger font and underlined. We note that the major cleavage product migrated in the gel as a 12 nt RNA in this gel electrophoresis system. (b) Quantification of the amount of uncleaved RNA over a period of 1 h. The results from the gel image were quantified by use of a phosphorimager and plotted. T50 and T70 represent the time required to cleave 50% and 70% of substrate, respectively. (c) Analysis of the effects of ribose and phosphodiester modifications on the kinetics of Nsp15 cleavage. The sequence of unmodified R16.4 is shown in its entirety. For the other RNAs, only the residues surrounding the modifications are shown. m denotes a 2′-methoxy group and f denotes a 2′-fluoro group. p denotes a phosphodiester and s denotes a phosphorothioate. T50 and T70 values for each RNA were derived from plots of the amount of products formed over a 1 h time-course.
Figure 2
Figure 2
Cleavage 3′ of uridylate. (a) R16.4, untreated (upper panel) and treated with Nsp15 (lower panel). The spectra show both the singly ionized product and the doubly ionized product, which is half of the mass of the former. The expected mass of untreated R16.4 is 5150.2 Da and cleavage 3′ of the U13 position should generate products of 4210.6 Da and 939.6 Da. (b) Cleavage of pCpUpA is inhibited by a modification in the uridine ribose 2′-OH. The oligonucleotide substrates were radiolabeled at its 5′ phosphate group using phage T4 polynucleotide kinase reactions containing [γ-32P]ATP. The time when the reaction was terminated is given in minutes. The presence of no enzyme (ϕ), Nsp15 or RNase A is noted above the corresponding reaction on the image of the TLC plate. The identities of the cleavage products are labeled to the right of the TLC image.
Figure 3
Figure 3
Effects of nucleotides adjacent to U13 on Nsp15 activity. (a) Gel images of the products generated by Nsp15 from RNA R16.4 and RNAs with altered residues immediately adjacent to U13. The sequences of nucleotides 12–14 of the RNAs tested are shown above the gel images. The residues altered from R16.4 are underlined. (b) Gel image showing an effect of nucleotides immediately upstream of –UC- on RNA cleavage by Nsp15. R16.4 with C14 was further modified to contain C, G or an A at the 12th position, indicated above the panel. Kinetic parameters, Km and Vmax, are indicated at the bottom of the image. (c) MALDI-TOF spectra of untreated (upper panel) and Nsp15 treated (lower panel) R16.4 containing the –CUC- sequence at positions 12–14.
Figure 4
Figure 4
Effects of base modifications on Nsp15 cleavage. (a) A summary of the uracil analogs tested in the context of RNA R16.4. The arrows denote the names of the RNAs with a modified uracil. T50 and T70 values of several RNAs with modified uridine bases at position 13 were derived from kinetic assays as described for Figure 1. (b) A structure of a highly conserved RNA present at the 3′-untranslated region of the SARS-CoV RNA named s2m. The schematics of the RNA are drawn from the information given by Robertson et al. (c) Cleavage of S2m by Nsp15 over a time-course. RNAs of 26 nt and 15 nt radiolabeled with phage T4 kinase and [32P]ATP were used as molecular mass markers. The denaturing 15% polyacrylamide gel on the top was electrophoresed using conditions that better resolve the longer RNAs while the denaturing 25% polyacrylamide gel at the bottom retained the smaller molecular cleavage products generated from the 5′ end of s2m.
Figure 5
Figure 5
Examinations of Nsp15 interaction with RNA analogs. (a) A real time endoribonuclease assay for Nsp15. The sequence of the quenched fluorescent substrate named rU (IDT, Coralville, IA) is shown, with fluorophores FAM and tetramethylrhodamine. The only ribonucleotide in this construct, uridylate, is in a larger font and underlined. A second version in which the uridylate was replaced with ribocytidine (rC) is used as a control. The fluorescent outputs from the two substrates were determined in the presence and in the absence of Mn2+, as indicated to the right of the graph. (b) A graph demonstrating inhibition of fluorogenic rU substrate turnover in the presence of increasing concentration of unlabeled rU (no inhibitor), a 16 nt DNA(D16.4), or R16.4 analogs (PT16 and N3meU). The equation used for the curve fit is in the box where the effects of various treatments are summarized: m2 denotes the apparent Km. (c) Image of an SDS/polyacrylamide gel where kinased RNAs (the identities of which are indicated above each lane) were crosslinked at 1200 μJ for 3 min to 1 μg of Nsp15. BSA was present at the same concentration. The letter ϕ is used to indicate that no protein was added in that reaction.
Figure 6
Figure 6
Nsp15-RNA interaction. (a) Inhibition of rU cleavage assay by RNAs rA16 or rU16. Increasing amounts of competitor RNAs (rA16 or rU16) were added to the reactions, allowed to equilibrate for 10 s and monitored for rate of cleavage. (b) Image of an SDS/polyacrylamide gel where kinased RNAs (identities of which are indicated above the lanes) were UV-crosslinked as described above to 500 ng of either WT or H234A mutant proteins. The mix contained an equal amount of BSA as a control for non-specific crosslinking. (c) Analysis of H234A-RNA interaction using fluorescence anisotropy. The anisotropy change was measured with increasing concentrations of H234A. Each datum point represents the average of ten anisotropy values. The Kd for the interaction and Hill coefficient (H.C) were derived from the binding isotherm using the Hill equation, as described in Materials and Methods. (d) SDS-PAGE image of radiolabeled rU16 crosslinked to H234A in the presence of increasing concentrations of MnCl2.
Figure 7
Figure 7
Analysis of Nsp15-RNA complex. (a) Gel-filtration profile of the catalytically inactive Nsp15 mutant, H249A in the presence (unbroken line) or in the absence (broken line) of biotin-labeled RNA U10. The positions where hexamer, trimer and monomer elute were characterized by Guarino et al. (b) Individual gel-filtration fractions on SDS-PAGE. (c) Slot blot of gel filtration fractions obtained when Nsp15 and RNA subjected to UV crosslinking were passed through the column. (d) Slot blot analysis of gel-filtration fractions performed with only RNA. Fraction numbers are indicated above the gel and blots. The RNAs in the blots were detected by probing with horse-radish peroxidase-conjugated streptavidin and developed using enhanced chemiluminiscence.
Figure 8
Figure 8
Cryo-EM 3D reconstruction of Nsp15 K289A. (a) Close to focus (1.5 μm) image of Nsp15. (b) Far-defocus (5.5 μm) image of the same area. (c) Refinement process monitored by Fourier shell correction. EOTEST is shown as a thick line. (d) The asymmetric triangle shows the orientation of the particles. (e) Upper row: class average of the particles; bottom row: reprojection from the final 3D model. (f) Surface display of the 3D structure, showing the six subunits in different colors. The figures to the left and right are the top and side views of the hexamer.
Figure 9
Figure 9
RNA binding by Nsp15 K289A. (a) Electron micrograph of negatively stained 2D crystal of Nsp15. (b) Fourier transform of the 2D crystal. (c) The 2D projection of Nsp15 with P2 symmetry. One unit cell is labeled;(a = 92.9 Å, b = 186.7 Å, γ = 87.6°). (d) The 2D projection of Nsp15- in complex with U16 (P2 symmetry, a = 95.8 Å, b = 179.8 Å, γ = 83.8°). (e) Two rU16s are fitted into the grooves present in the interface between the dimers of trimers of a hexameric Nsp15. The bottom image is rotated by 90° relative to the top image.
Supplementary Figure S1
Supplementary Figure S1
Kinetic analysis of cleavage at the uridylate in RNA R16.4 by Nsp15. Shown are the results from the three independent experiments in which the parameters were determined. Each experiment consisted of nine RNA concentrations analyzed over five time points. The mean Km and Vmax values derived from these experiments are summarized in Fig. 1B.
Supplementary Figure S2
Supplementary Figure S2
Comparison of the structures of the negatively stained Nsp15 hexamer and the cryo-EM structure filtered to 2 nm resolution. (A) Ttop and side views of the negatively-stained Nsp15 K289A. (B) Top and side views of the cryo-EM structure of Nsp15 K289A.
Supplementary Figure S3
Supplementary Figure S3
Co-crystals of Nsp15 K289A mutant protein without (A) and with RNAs (B, C). SLT is an tightly folded RNA of 35-nt (Kim et al., 2001) while poly(I:C) is a chemically synthesized RNA of 40 bps. The boxes in each panel define a unit cell. Note that the structures of each of the protein differed with the RNA bound, suggesting that Nsp15 could adjust its conformation in response to the RNA.
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