Spontaneous base flipping helps drive Nsp15’s preferences in double stranded RNA substrates

Figures

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  Fig. 1. U’s complement affects its susceptibility to cleavage by Nsp15. Unpaired U is especially susceptible, while U•G is especially resistant. A dsRNA oligo substrate design. The target strand, containing U19 (marked with *) is labeled with 5′ Cy5 and 3′ Fl. Us in the target strand other than U19 have been substituted for 2′-F-U (light gray) which is not cleavable by Nsp15. The complement strand is unlabeled. Five substrates were generated with this design, changing only the nucleotide complementary to U19 (unpaired U, U-A, U•G, U•U’, or U•C). B Representative denaturing PAGE gels of timecourse nuclease assays for each of the five substrates, performed at 50 nM Nsp15 and 500 nM dsRNA. Uncleaved RNA appears in both Cy5 (red) and Fl (blue) channels, resulting in a pink signal in the overlaid image. C Percent of target strand cleaved over time, quantified via the intensity of the uncleaved RNA band and normalized to the 2 min timepoint. Each point with error bars represents average and standard deviation for three independent reactions, each using a distinct protein preparation (N = 3 biological replicates). Images of each gel and quantification data are provided in the Source Data file.

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  Fig. 2. Nsp15 prefers unpaired U over paired and mismatched Us, even in substrates without 2′-F modification. A Substrate design. The target strand is labeled with 5′ Cy5 and 3′ Fl, and contains 11 cleavable Us. The complement strand is unlabeled. Three substrates were created with this design by changing the nucleotide complement to U14 to produce either a U-A, U•C, or unpaired U. B Representative denaturing PAGE gels of time-course nuclease assays for each of the three substrates. The U-A and U•C substrates show similar patterns of cleavage with Us in the 4 positions nearest the ends being most favored. The unpaired U substrate shows a different pattern, with cleavage at the unpaired U14 outpacing cleavage at all other Us. Note the near-complete disappearance of the target strand by 30 min, approximately twice as fast as in U-A and U•C substrates. Three independent reactions (each using a distinct protein preparation) were performed with each substrate. Images of each gel are provided in the Source Data file.

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  Fig. 3. Substrate containing highly favored unpaired U enables reconstruction of Nsp15-bound dsRNA with defined sequence. A Design of dsRNA oligo incubated with Nsp15 for cryo-EM experiments; identical to unpaired U substrate used in Fig. 2. Nucleotides that were found to be sites of interaction with Nsp15 are labeled with their position number. Labels in the target strand containing the unpaired U are positive, with position 1 at the 5′ end; labels in the complement strand are negative, with position -1 at the complement strand’s 5′ end. B Cryo-EM density map of dsRNA-bound Nsp15 (H235A mut) at a resolution of 3.24 Å, including dsRNA (pink), protomers of Nsp15 that interact with dsRNA (P1, P2, P4 in shades of dark teal), and remaining protomers of Nsp15 (P3, P5, P6 in shades of light blue-gray). C Closeup view of atomic model and mesh cryo-EM density map showing dsRNA residues surrounding the flipped-out U14, which is engaged in the active site of Nsp15 protomer P1. H-bonds are shown in cyan. D Cryo-EM density map of Nsp15-bound dsRNA, colored by local resolution. Protomer P1 is outlined in black. E Residues of Nsp15 protomer P1 calculated (via dr_sasa47) to interact with dsRNA, with P1…

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  Fig. 4. A U’s susceptibility to cleavage by Nsp15 parallels its tendency to spontaneously flip. A dsRNA substrate design, with observable target U* (in black). For cleavage assays, U* = 2′-OH, non-target Us in target strand = 2′-F-U. Target strand has 5′ Cy5 and 3′ Fl labels, as in Fig. 1. For NMR, U* = 2′-F-U, all other Us = 2′-OH. From this design, a set of substrates were created by changing 2 nt on both 5′ and 3′ sides of U* (gray, marked with?) to different sequences, shown in (B). For each sequence, U* was either unpaired or engaged in a U•A pair (orange). C Percent of target strand cleaved over time, quantified via intensity of the uncleaved RNA band and normalized to the 2 min timepoint. Each point with error bars represents average and standard deviation for at least three independent reactions, each using a distinct protein preparation (N = 3 biological replicates). Images of each gel and quantification data are provided in the Source Data file. Data for substrate 1 (“AAUUU”) in panel C is reproduced from Fig. 1C for clarity and ease of comparison. D 1D 19F NMR spectra for each dsRNA substrate in the absence of Nsp15. Each peak is labeled with its shift (ppm, top value) and…

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  Fig. 5. Nsp15 prefers to cleave unpaired U in 1 nt bulge in stem loop RNA. A Design of substrates with sequence from the SARS-CoV-2 genome’s Stem Loop 4 (SL4) used for 19F NMR. Key positions are marked where U was substituted for 2′-F-U to enable 19F NMR. Nts are labeled according to position in the positive strand viral genomic RNA. B Overlay of top ten structures of SL4 determined by Vögele et al.52, with key Us color coded (PDBID 8CQ1). Note that U95 is consistently flipped out, while U104 and other Us are consistently stacked. C 1D 19F NMR spectra of five different SL4 derivatives, each with a single 2′-F-U at the indicated position. Each peak is labeled with its shift (top value) and linewidth (bottom value, in parentheses). D Design of SL4 substrate used for enzyme assay, with 5′ Cy5 and 3′ A4-Fl. E Representative denaturing PAGE gel of time-course nuclease assays, with Cy5 (red) and Fl (blue) channels separated to facilitate band identification. Three independent reactions (each using a distinct protein preparation) were performed with each substrate. Images of each gel are provided in the Source Data file. F Mass spectrum showing multiply charged ions representing the major…

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  Fig. 6. Proposed mechanism for conformational capture of flipped U in dsRNA by Nsp15. Bases can spontaneously flip towards either the major or minor groove of dsRNA. Nsp15 can only interact with key functional groups of the U if the U has flipped into the minor groove; these interactions are hindered when the base flips into the major groove, preventing cleavage. RNA is modeled from PDBs 7JL3, 8CQ1, and our model 9BIH.