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The structure of the box C/D enzyme reveals regulation of RNA methylation

Nature volume 502, pages 519–523 (2013)Cite this article

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Abstract

Post-transcriptional modifications are essential to the cell life cycle, as they affect both pre-ribosomal RNA processing and ribosome assembly. The box C/D ribonucleoprotein enzyme that methylates ribosomal RNA at the 2′-O-ribose uses a multitude of guide RNAs as templates for the recognition of rRNA target sites. Two methylation guide sequences are combined on each guide RNA, the significance of which has remained unclear. Here we use a powerful combination of NMR spectroscopy and small-angle neutron scattering to solve the structure of the 390 kDa archaeal RNP enzyme bound to substrate RNA. We show that the two methylation guide sequences are located in different environments in the complex and that the methylation of physiological substrates targeted by the same guide RNA occurs sequentially. This structure provides a means for differential control of methylation levels at the two sites and at the same time offers an unexpected regulatory mechanism for rRNA folding.

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Figure 1: sR26 RNA in the P. furiosus box C/D complex.
Figure 2: Non-equivalent fibrillarin environments in the apo-box C/D sRNP.
Figure 3: Catalytic structure of the box C/D sRNP.
Figure 4: Methylation of substrate D and D′ is sequentially regulated.
Figure 5: Regulatory mechanism for sequential methylation.

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Protein Data Bank

Data deposits

Coordinates of the holoenzyme have been deposited in the Protein Data Bank under accession code 4BY9.

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Acknowledgements

This work was supported by DFG grant CA294/3-1, by EU FP7 ITN project RNPnet (contract number 289007) and by the EMBL. For the SAXS and SANS experiments we thank the ESRF and ILL, BAG MX1219 and the D22 BAG system for beam-time as well as A. Martel and P. Pernot for help with the setup of the instruments. We thank J. Wöhnert for the gift of 13C-labelled SAM and C. Kreutz for the gift of [13C]2′OCH3 substrate RNA. We thank John Kirkpatrick for the assignment of the L7Ae resonances.

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Authors and Affiliations

  1. European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstrasse 1, D-69117 Heidelberg, Germany,

    Audrone Lapinaite, Bernd Simon, Lars Skjaerven, Magdalena Rakwalska-Bange & Teresa Carlomagno

  2. Université Grenoble Alpes, Institut de Biologie Structurale, F-38027 Grenoble, France,

    Frank Gabel

  3. Commisariat à l’Energie Atomique, Direction de Sciences du Vivant, Institut de Biologie Structurale, F-38027 Grenoble, France,

    Frank Gabel

  4. Centre National de la Recherche Scientifique, Institut de Biologie Structurale, F-38027 Grenoble, France,

    Frank Gabel

  5. Institut Laue-Langevin, F-38042 Grenoble, France,

    Frank Gabel

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  1. Audrone Lapinaite
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Contributions

A.L., B.S., F.G. performed experiments and analysed data; M.R.-B. prepared the samples; L.S. and B.S. performed calculations; T.C. designed the project, analysed data and wrote the manuscript together with A.L.

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Correspondence to Teresa Carlomagno.

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Extended data figures and tables

Extended Data Figure 1 Overview of the components, structural models and biochemical characterization of the box C/D RNP.

a–c, Amino acid sequences of the proteins Nop5 (a), fibrillarin (b) and L7Ae (c) of Pyrococcus furiosus with secondary structure annotation. Helices, yellow bars; β-sheets, blue arrows. d, Schematic representation of the box C/D sRNA (in archaea) or snoRNA (in eukaryotes). The box C/C′ and D/D′ are underlined in green and yellow, respectively. The substrate D (D′), which binds to the guide sequence upstream of box D (D′), is in firebrick red (salmon). A star marks the nucleotides that are subject to methylation. e, Sequences of the asymmetric sR26 RNA (asR26) from Pyrococcus furiosus and of the partially symmetrized version of the sR26 RNA (ssR26) used in this structural study. The asR26 differs from the wild-type sequence in that three base pairs have been added to the terminal helix to increase stability. f, Model of mono-RNP from 3PLA.pdb16,17. The complex consists of one molecule of sRNA bound to two L7Ae molecules. Two Nop5 copies dimerize with the axis of the coiled-coil domains almost parallel to the sRNA long axis. The two fibrillarin copies are in contact with the substrate RNAs (D and D′), which occupy identical environments. g, Model of di-RNP from refs 17, 18 (3NMU.pdb and electron microscopy envelope emd_1636). Two molecules of sRNA (in yellow) occupy two sides of a square-shaped structure and are perpendicular to the coiled-coil dimerization domains of two Nop5 dimers (grey). The four fibrillarin copies (blue) are in the ‘off’ position. h, Size exclusion chromatography elution profile of the fully assembled apo-box C/D sRNP. The elution volume corresponds to a complex of ∼400 kDa in size, as extrapolated from the elution profile of the standards mixture (green, molecular weight (MW) in kDa is indicated for each peak). The peak around 150 kDa corresponds to the excess of the dimeric Nop5–fibrillarin complex used for the assembly of the box C/D sRNP. i, Native gel of the apo- (lane –) and holo- (lane +) box C/D sRNP. The lane M was loaded with standard proteins of different MW (in kDa). The electrophoretic profile of both the apo- and holo-complex is consistent with a particle of ∼400 kDa MW, as expected for the di-RNP.

Extended Data Figure 2 Verification of interaction interfaces in the sub-complexes.

a, Overlap of the 13C–1H correlations of free fibrillarin (orange) and fibrillarin in the Nop5–fibrillarin complex (blue). b, Methyl groups of fibrillarin with 13C–1H chemical-shift perturbations > 0.1 upon transition from the free protein to the apo-box C/D complex are plotted on the structure of the Nop5-NTD–fibrillarin sub-complex from 2NNW.pdb (pink spheres). The location of the chemical-shift perturbations confirms that the interaction surface between fibrillarin and Nop5 in the box C/D di-RNP is conserved with respect to previously determined crystal structures of the Nop5–fibrillarin sub-complex. c, The chemical-shift perturbations, calculated according to equation (3), are plotted against the protein sequence (blue) and compared with chemical-shift perturbations observed for the Nop5-NTD–fibrillarin complex (grey). The high similarity between the two chemical-shift perturbations sets confirms that in the apo-box C/D RNP the fibrillarin interacts exclusively with the Nop5-NTD. d, PRE effects measured for fibrillarin upon paramagnetic labelling of the Nop5(E68C) mutant (blue), compared to those predicted from the structure of the Nop5–fibrillarin sub-complex in 2NNW.pdb (red). The good agreement of the data further confirms that the interaction mode between the Nop5 and fibrillarin is conserved. Similar agreement is obtained for the paramagnetically labelled Nop5(E97C) mutant. An equivalent analysis confirms the interaction surface between L7Ae and the Nop5-CTD.

Extended Data Figure 3 Structure calculation protocol.

a, Sub-complexes whose structure was considered as preserved in the full box C/D RNP. From left to right, L7Ae–K-turn-RNA–Nop5-CTD from 3NMU.pdb, Nop5-NTD–fibrillarin and Nop5-CC domain from 3NMU.pdb, model of the guide–substrate RNA A-form helix. b, Points of flexibility to allow assembly of the full box C/D RNP. Left, the K-turn RNA and guide–substrate RNA duplex elements are allowed to reorient with respect to each other hinging on the flexible bonds of A14, A26, A50, A71 (boxed in pink). Right, the three domains of the Nop5 protein are allowed to reorient with respect to each other hinging on the flexible amino acid stretches highlighted in red. c, The position of the spin labels is shown on the experimental structure of the apo-complex as red spheres, with the blue (fibrillarin) or green (L7Ae) funnels indicating the protein for which PRE effects were measured.

Extended Data Figure 4 SAS analysis of the apo- and holo-complexes.

Experimental small-angle neutron (SANS) and X-ray (SAXS) scattering curves for the apo- (left) and holo- (right) box C/D complex with their respective fitting to the curves back-calculated from the structure of Fig. 2 and Fig. 3c. The quality of the fitting of each experimental curve to the theoretical one is given next to each curve (χ2). The insets represent the Guinier fits. Several curves have been artificially scaled for visualization purposes. a, The SANS data are for samples of the apo-box C/D RNP assembled from [2H]RNA and [1H]proteins (dRNA, yellow); [2H]Nop5, [1H]L7Ae, [1H]fibrillarin and [1H]RNA (dNop5, black), [2H]fibrillarin, [1H]L7Ae, [1H]Nop5 and [1H]RNA (dFib, blue); [2H]L7Ae, [1H]Nop5, [1H]fibrillarin and [1H]RNA (dL7Ae, green); [2H]fibrillarin and [2H]RNA, [1H]L7Ae and [1H]Nop5 (dRNAdFib, purple). All complexes were measured in 42%/58% D2O/H2O. b, The SANS data are for samples of the holo-box C/D RNP assembled from [2H]RNA and [1H]proteins (dRNA, yellow); [2H]Nop5, [1H]L7Ae, [1H]fibrillarin and [1H]RNA (dNop5, black), [2H]fibrillarin, [1H]L7Ae, [1H]Nop5 and [1H]RNA (dFib, blue). Two sets of experimental data are shown for each sample, after addition of one equivalent of substrate (low substrate RNA (subRNA), half-loaded complex) and after saturation of all substrate-binding sites (high subRNA, holo-complex). The half-loaded complex shows a similar shape as the holo-complex, indicating that the large conformational change observed upon transition from the apo- to the holo-form occurs already after half binding sites have been occupied. The data of the holo-complex slightly deviate from the back-calculated curves due to strong inter-particle repulsion, as verified by repeating the experiments at different concentrations. c, SANS and SAXS scattering curves of the apo-box C/D sRNP assembled either with all [1H]components (allH SANS, light blue and SAXS, red) or with [2H]fibrillarin and [2H]RNA, [1H]L7Ae and [1H]Nop5 (dRNAdFib, purple). All complexes were measured in 100% H2O. d, SAXS scattering curve of the holo-box C/D sRNP assembled with all [1H]components and after saturation of all substrate-binding sites. e, Fit of the experimental curves from dRNA (SANS, yellow), dNop5 (SANS, black), dRNAdFib (SANS, purple) and allH (SAXS, red) apo-complex samples with the scattering curves of the apo-complex structure calculated here (bold lines) or of the model proposed in ref. 17 and schematically shown in Extended Data Fig. 1g (thin lines). The latter model displays a poor fit to the data and can be considered invalid under our experimental conditions. f, Fit of the experimental curves from dRNA (SANS, yellow), dNop5 (SANS, black) and allH (SAXS, red) holo-complex samples with the scattering curves of the holo-complex structure calculated here (bold lines) or the structure of the mono-RNP proposed in ref. 16 (3PLA.pdb) and schematically shown in Extended Data Fig. 1f (thin lines). The latter model displays a poor fit to the data and can be considered invalid under our experimental conditions. g, Kratky plot representations (I(Q)Q2) of dFib and dNop5 SANS data at 42%/58% D2O/H2O and of the SAXS data of the apo- (continuous lines) and holo- (dashed lines) complex, illustrating the conformational change occurring upon substrate binding. h, Fit of the experimental SANS curve from the allH apo-complex to the scattering curves generated from the apo-complex structure calculated here (light blue) and from the electron-microscopy-derived envelopes of three other apo-sRNP17,18. The SANS curves were calculated from the deposited electron microscopy densities using the ATSAS programs EM2DAM and CRYSON in default parameters by applying the contour levels proposed in the electron microscopy databank. The electron microscopy envelopes do not display a good fit to the SANS data and clearly differ among each other.

Extended Data Figure 5 Ensemble representation of the structures of the box C/D sRNP in the absence or presence of target RNA.

a, Ensemble of structures representing the apo-box C/D sRNP. The structures have been superimposed on the CC domains of the Nop5 proteins. RNA, red; L7Ae, green; Nop5, grey; fibrillarin, blue. The average pair-wise r.m.s.d. to the structure closest to the mean, calculated for the Cα atoms of all proteins excluding the Nop5-NTDs and fibrillarin copies, is 4.8 Å (Extended Data Table 2). b, Ensemble of structures representing the holo-box C/D sRNP. The structures have been superimposed on the CC domains of the Nop5 proteins. RNA, red; L7Ae, green; Nop5, grey; fibrillarin, blue. The average pair-wise r.m.s.d. to the structure closest to the mean, calculated for the Cα atoms of all proteins excluding the Nop5-NTDs and fibrillarin copies not in contact with the RNA, is 5.2 Å (Extended Data Table 2).

Extended Data Figure 6 Box C/D sRNP in the absence of substrate RNA (apo-complex).

a, Structure of the apo-box C/D sRNP with numbering. The protein copies numbered 1 (2) and 3 (4) are associated with the box C′/D′ (C/D) elements of two different sRNA molecules. Nop5 copies 1 and 3 are in surface representation (light grey and pale blue, respectively) and occupy the centre of the particle. The guide D and D′ sequences are coloured firebrick and salmon, respectively. The rest of the RNA is in yellow. The CC domains of Nop51 (Nop53) and Nop52 (Nop54) dimerize. b, Each of the two ssR26 RNA molecules is sandwiched between positively charged surfaces of the Nop5-CTDs recognizing the box C/D (below) and box C′/D′ (above) elements.

Extended Data Figure 7 NMR analysis of fibrillarin in different environments and PRE analysis of the apo- and holo-sRNPs.

a, Overlap of the 13C–1H correlations of fibrillarin in the Nop5–fibrillarin complex (cyan) and in the apo-box C/D sRNP (blue). The chemical shifts of ILV methyls are virtually identical, indicating that in the apo-complex the fibrillarin is in the ‘off’ position and experiences a similar environment as in the Nop5–fibrillarin sub-complex. b, Overlap of the 13C–1H correlations of the free fibrillarin ILV methyl groups upon titration of duplex RNA (0, 0.5, 1 molar equivalents). The absence of chemical-shift perturbations indicates that free fibrillarin does not bind to the duplex RNA; however, when this structure is present in the complex, fibrillarin binds to it (Fig. 3a). c, Overlap of the 13C–1H correlations of the fibrillarin ILV methyl groups in the apo-box C/D enzyme (blue) and after titration of C9-2′OCH3 substrate D′ (pink). The absence of chemical-shift perturbations indicates that fibrillarin in the complex does not bind to the guide–substrate duplex when the substrate is 2′-O-methylated, whereas it binds to the non-methylated duplex (Fig. 3a). The resonances that split in two sets in Fig. 3a are assigned. d, Paramagnetic relaxation enhancement (PRE) data for the apo- (left) and holo- (right) box C/D complexes. Only three representative data-points are shown. Experimental points are in blue, whereas the red points are back-calculated from the structures. Assignments are shown next to the corresponding values for some of the methyl groups. The apo-complex data for the Nop5(E68C) mutant show some deviations for L7Ae residues 11, 15, 52, 53, 75, 78. The PRE effects in this region mimic the PRE effects of the holo-complex; this indicates occasional fly-casting motions of fibrillarin that bring the methyltransferase close to the RNA already in the apo-complex. These motions might represent the mechanism through which fibrillarin recognizes the substrate RNA in the holo-complex. The flexibility of fibrillarin in the apo-complex is also demonstrated by the residual deviations of the experimental PRE data from the back-calculated ones for the Nop5(D250C) mutant.

Extended Data Figure 8 Box C/D sRNP in the presence of substrate RNA (holo-complex).

a, Structure of the holo-box C/D sRNP with numbering. The protein copies numbered 1 (2) and 3 (4) are associated with the box C′/D′ (C/D) elements of two different sRNA molecules. Nop5 copies 1 and 3 are in surface representation (light grey and pale blue, respectively); their CTDs and NTDs occupy the centre of the particle. However, the NTDs of Nop51 and Nop53 are swapped with respect to the CTDs, in such a way that the Nop51-NTD comes close to the Nop53-CTD and vice-versa. This domain swap causes Fib3 to methylate the substrate sequence close to L7Ae1 and Nop51-CTD. The guide–substrate D and D′ helices are coloured firebrick and salmon, respectively. The rest of the RNA is in yellow. The CC domains of Nop51 (Nop53) and Nop52 (Nop54) dimerize. b, The CTDs of Nop51 and Nop53 interact with each other at the centre of the complex. The contact occurs via the α6, α10 and α12 helices and loop α10–α11, which show mutual charge and shape complementarities. Shown in the figure are the two Nop5-CTDs in contact with each other, one in surface and one in transparent cartoon representation. The black line connects the contact areas of the two protein copies. c, Structural basis for the specificity of methylation. The fibrillarin in contact with the substrate RNA (Fib3, blue surface) is sandwiched between the L7Ae connected with the same box C′/D′ element (L7Ae1, green surface) and the Nop5-CTD connected with the box C/D element of the same RNA molecule (Nop54-CTD, dark grey surface). Both the L7Ae and the Nop5-CTD hinder sliding of fibrillarin along the guide–substrate duplex and direct it to the fifth nucleotide downstream of box D′. d, Details of the contacts of Fib3 (in cyan) with the Nop54-CTD and L7Ae1 (both in electrostatic surface representation). Three sequential aspartic acid amino acids (D17–D19) from the N-terminal tip of fibrillarin lock the methyltransferase to a positively charged surface of the Nop54-CTD (composed by the tips of helix α10, α11 and loop α10–α11). On the opposite side, the α3-α4 surface of fibrillarin is in electrostatic contact with helix α3 of L7Ae. Amino acids of fibrillarin shown in ball and sticks are within 5 Å from L7Ae.

Table 1 Extended Data Table 1
Full size table
Table 2 Extended Data Table 2
Full size table

Supplementary information

Structure of the holo-Box C/D RNP

The molecular components are colored as in Figure 3 and Extended Data Figure 8. After 18 seconds the video zooms on one of the Fibrillarin copies, bound to the guide-substrate duplex, and shows that the Fibrillarin position is confined by packing with L7Ae and Nop5-CTD. The interaction of Fibrillarin amino acids D17-19 with the positively charged surface of Nop5-CTD is also shown (Extended Data Figure 8d). After 42 seconds the video zooms on the centre of the particle and shows the mutual interaction of two Nop5-CTDs via charge complementarities (Extended Data Figure 8b). (MOV 54011 kb)

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Lapinaite, A., Simon, B., Skjaerven, L. et al. The structure of the box C/D enzyme reveals regulation of RNA methylation. Nature 502, 519–523 (2013). https://doi.org/10.1038/nature12581

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