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. Author manuscript; available in PMC: 2013 Feb 14.
Published in final edited form as: Mol Cell. 2010 Sep 24;39(6):939–949. doi: 10.1016/j.molcel.2010.08.022

Structural basis for substrate placement by an archaeal box C/D ribonucleoprotein particle

Song Xue 1,*, Ruiying Wang 2, Fangping Yang 2, Rebecca M Terns 3, Michael P Terns 3, Xinxin Zhang 4, E Stuart Maxwell 4, Hong Li 1,2
PMCID: PMC3572848  NIHMSID: NIHMS232133  PMID: 20864039

Abstract

Box C/D small nucleolar and Cajal body ribonucleoprotein particles (sno/scaRNPs) direct site-specific 2’-O-methylation of ribosomal and spliceosomal RNAs and are critical for gene expression. Here we report crystal structures of an archaeal box C/D RNP containing three core proteins (fibrillarin, Nop56/58, and L7Ae) and a halfmer box C/D guide RNA paired with a substrate RNA. The structure reveals a guide-substrate RNA duplex orientation imposed by a composite protein surface and the conserved GAEK motif of Nop56/58. Molecular modelling supports a dual C/D RNP structure that closely mimics that recently visualized by electron microscopy. The substrate-bound dual RNP model predicts an asymmetric protein distribution between the RNP that binds and that methylates the substrate RNA. The predicted asymmetric nature of the holoenzyme is consistent with previous biochemical data on RNP assembly and provides a simple solution for accommodating base-pairing between the C/D guide RNA and large ribosomal and spliceosomal substrate RNAs.

Box C/D sno/scaRNAs (small RNAs, or sRNAs in archaea) are among the many noncoding RNAs that guide enzymatic reactions in Eukarya and Archaea (Hannon et al., 2006; Matera et al., 2007). The large majority of box C/D RNAs guide site-specific 2’-O-methylation of ribosomal and spliceosomal RNA (Decatur and Fournier, 2003; Fatica and Tollervey, 2002; Kiss-Laszlo et al., 1996; Kiss, 2002; Terns and Terns, 2002; Weinstein and Steitz, 1999) while a small subset including the essential U3, U14, and the vertebrate U8 RNAs also guide site-specific cleavage of ribosomal RNA (Kass et al., 1990; Liang and Fournier, 1995; Peculis and Steitz, 1993; Savino and Gerbi, 1990; Venema and Tollervey, 1995). In RNA-guided processes, the RNA generally recognizes and secures the substrate RNA while partner proteins catalyze the actual chemical reaction. Regardless of type, box C/D RNAs function with a minimal set of 3-4 core proteins (Caffarelli et al., 1998; Gautier et al., 1997; Lafontaine and Tollervey, 1999; Lafontaine and Tollervey, 2000; Lyman et al., 1999; Omer et al., 2002; Schimmang et al., 1989; Tyc and Steitz, 1989; Watkins et al., 1998; Wu et al., 1998). These include fibrillarin (Nop1 in yeast), Nop56, Nop58, and 15.5kD (in yeast, Snu13p and in archaea, L7Ae). A single homologue of Nop56 and Nop58, Nop56/58, is found in archaea. There is solid evidence that fibrillarin is the catalytic subunit responsible for the methyl transfer reaction (Aittaleb et al., 2003; Aittaleb et al., 2004; Tollervey et al., 1993)

A key issue in the mechanism of RNA-guided methylation is how the large and complex rRNA or snRNA substrates access the enzyme active site through pairing with the highly restricted guide RNA. Box C/D RNAs possess a core structure that comprises an internal loop flanked by two similar box C/D and C’/D’ motifs, collectively referred to as “the box C/D module” (Figure 1A). The internal loop includes 10-21 nucleotides that are complementary to up to two different substrate RNAs (Bachellerie and Cavaille, 1998). The box C/D module is conserved and contains two hallmark sequence motifs of AUGAUG (box C or C’) and CUGA (box D or D’) (Kiss-Laszlo et al., 1998). The combined box C (C’) and D (D’) sequences form a conserved RNA structural motif, called the kink-turn (K-turn), around which the RNP proteins assemble. Snu13p/15.5kD/L7Ae binds specifically to the K-turn (Kuhn et al., 2002; Szewczak et al., 2002; Watkins et al., 2000). This is then followed by binding of Nop56/58 or the Nop56/58-fibrillarin complex (Omer et al., 2002; Rashid et al., 2003; Watkins et al., 2002). The K-turn-L7Ae or K-turn-15.5kD interaction has been visualized at atomic resolution (Charron et al., 2004; Hamma and Ferre-D’Amare, 2004; Moore et al., 2004; Oruganti et al., 2005; Vidovic et al., 2000). Structures of the isolated Nop56/58-fibrillarin complex (Aittaleb et al., 2003; Oruganti et al., 2007) and its complex with the K-turn-L7Ae (Ye et al., 2009) are also available. These structural data lead to a bipartite RNP model in which a single hairpin box C/D RNA is clamped at each end by a set of Nop56/58-fibrillarin-L7Ae proteins leaving the internal loop exposed for interaction with substrate RNAs (Aittaleb et al., 2003; Oruganti et al., 2007; Ye et al., 2009). However, the bipartite model is met with several challenges. First, the length of an intact box C/D RNA is estimated to be shorter than the distance between the two K-turns in the complex, which is established by the long coiled-coil connector domains of Nop56/58. Second, it is expected to be topologically challenging for a large substrate RNA to form more than 10 base pairs with the central internal loop whose size is restricted by flanking proteins.

Figure 1.

Figure 1

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Schematics of the box C/D RNA and the overall structures of the substrate-bound halfmer RNP. A. Schematic drawing of a generic box C/D RNA and the model halfmer RNA used in crystallization. K-turn nucleotides are outlined in red and the substrate nucleotides are colored in yellow. The complex contains guide RNA nucleotides 1-34 and substrate RNA 1-12 or 1-13 for the two protomers respectively. B. Structure of the halfmer complex. Both protomers of Nop56/58 are colored green, fibrillarin is in blue, L7Ae is in cyan, guide RNA is in red and substrate RNA is in yellow. The strictly conserved Nop56/58 GAEK motif is highlighted in pink and SAM molecules are in magenta. “R face” denotes the side of RNA binding and “F face” denotes that of fibrillarin binding. C. Electron density (3Fo-2Fc) map at 1σ around the RNA complex. D. Close-up view of the RNA binding region.

The box C/D RNP methyltransferase employs a remarkable strategy to specify the site of modification of the substrate RNA, the physical basis of which is unknown. 2’-O-methylation takes place strictly on the substrate RNA nucleotide paired with the 5th nucleotide upstream of the box D or D’ (N+5 rule) (Cavaille et al., 1996; Kiss-Laszlo et al., 1996; Tycowski et al., 1996). Conservation of the N+5 rule in both Archaea and Eukarya places the catalytic site uniformly ~20 Å away from the first box D (or D’) nucleotide. Currently, none of the available structural models is able to provide a molecular mechanism responsible for the modification site specification owing to the lack of substrate-bound structures.

The traditional bipartite model, in which the pseudosymmetric box C/D RNA lies longitudinally along the Nop56/58-fibrillarin homodimer, has recently been challenged by the electron microscopic observations that a functional box C/D RNP comprises a pair of isologously (face-to-face) bound RNPs (diRNP) (Bleichert et al., 2009). The diRNP predicts that an intact box C/D RNA binds vertically to the Nop56/58-fibrillarin dimer and across two parallel Nop56/58-fibrillarin dimers (Bleichert et al., 2009). The remaining Nop56/58-fibrillarin halves are occupied by an additional C/D RNA, leading to a functional particle containing four sets of proteins and two box C/D RNA molecules. It was also suggested that the diRNP potentially contains four substrate binding sites, which is believed to be an efficient means for capturing substrate RNA (Bleichert et al., 2009). These predictions, however, could not be confirmed or refuted due to the lack of atomic RNP structures. The previously determined crystal structure of Sulfolobus solfataricus (Ss) Nop56/58-fibrillarin bound with L7Ae and a halfmer RNA, confirmed the site of K-turn binding on Nop56/58, but was ambiguous on locations of RNA guide region and fibrillarin owing to the lack of a bound substrate RNA and unwanted crystallographic packing interactions. The processes of RNP assembly and substrate binding, therefore, remain elusive.

Here we report crystal structures of a C/D RNP methyltransferase from the archaeon Pyrococcus furiosus (Pf) bound with a substrate RNA at 2.7 Å resolution. The structural complex comprises Pf L7Ae residues 4-124 (full length 124) Pf Nop56/58 residues 8-369 (full length 402), Pf fibrillarin residues 1-227 (full length 227), nucleotides 1-34 of a model box C/D RNA and nucleotides 1-12 of a 13mer substrate RNA (Figure 1A). We describe previously unknown interactions involving conserved protein elements in stabilizing the guide-substrate complex. We also describe construction of a substrate-bound diRNP model that is consistent with both the overall dimension and protein distribution of the electron microscopy model. Finally, we propose mechanisms of substrate binding and catalysis of box C/D RNPs that is consistent with mutational and previously known biochemical data.

Results

Overall structure of the substrate-bound RNP

All three RNP structures were determined by molecular replacement methods using the Pf Nop56/58-fibrillarin complex coordinates (PDBcode: 2NNW). The RNA and L7Ae protein could be modelled into the electron densities phased with the Nop56/58-fibrillarin complex. Details of the structure determinations are described in Experimental Procedures. The full complex contains two sets (protomers) of the three proteins and RNA that dimerize via the coiled-coil interactions between two Nop56/58 proteins. The 13mer substrate RNA is bound to the guide region of the two box C/D RNAs (Figure 1B, 1C, & 1D). Two protomers of both RNPs superimpose well with each other (0.82 Å RMSD for all Cα atoms). Specifically, the substrate-guide duplexes of the two protomers differ by only ~3° in their helical direction, suggesting that crystal packing had little or no impact on RNP formation. The overall structure and domain orientations of the individual RNP components of the full complex are similar to those of the isolated Pf Nop56/58-fibrillarin complex but differ from those of the Archaeoglobus fulgidus (Af) and Ss Nop56/58-fibrillarin complexes (Aittaleb et al., 2003; Ye et al., 2009). The key difference lies in the position of fibrillarin, which results from rotations of the N-terminal domain of Nop56/58 around a peptide hinge. In the orientation shown in Figure 1B, the long coiled-coil represents a meridian that divides the bound RNA (guide and substrate) and fibrillarin. The two box C/D RNA modules fall on one side of the meridian (R face) and the two fibrillarin are on the other (F face) (Figure 1B).

It is immediately apparent from Figure 1B that the substrate binds far away from the nearest fibrillarin catalytic subunit. Although it is tempting to conclude that the structure captures an incorrectly bound substrate due to the use of catalytically inactive halfmer box C/D RNA in crystallization, detailed analyses presented below instead suggests an unexpected mechanism of modification and that the observed bound substrate likely represents a productive binding mode.

Role of the GAEK motif in specific C/D RNP assembly

Sequence alignment of Nop56/58 proteins has identified a strictly conserved motif containing Gly-Ala-Glu-Lys tripeptide (GAEK motif), suggesting its important role in box C/D RNP function (supplementary material, Figure S1 and Aittaleb et al., 2003). This region, however, has never been observed in any previously determined RNA-free or RNA-bound complex structures owing to its disorder (Aittaleb et al., 2003; Oruganti et al., 2007; Ye et al., 2009). In the presence of a guide RNA or a guide RNA-substrate complex, the GAEK motif structure unveils its remarkable roles in RNP assembly and substrate binding.

Box C/D RNP methyltransferases exhibit multi-level assembly specificity. L7Ae (Snu13p/15.5kD) binds to the K-turn specifically and this interaction has been studied extensively in isolated L7Ae-RNA complexes (Charron et al., 2004; Hamma and Ferre-D’Amare, 2004; Moore et al., 2004; Oruganti et al., 2005). The C-terminal domain of Nop56/58 subsequently binds to a composite surface of L7Ae and K-turn and facilitates another key specific interaction. The α-helix, α9A (see secondary element labels in Figure S1) and its C-terminal extension containing the well-conserved GAEK motif (residues 293-312) (collectively termed GAEK motif) are wedged between the guide and the non-guide strands (Figure 2A). Although highly conserved, the entire GAEK motif is unstructured in all previously observed complexes involving Nop56/58-fibrillarin complexes (Aittaleb et al., 2003; Oruganti et al., 2007; Ye et al., 2009). The three terminal base pairs of the box C/D RNA are unwound by α9A and its C-terminal extension so both strands wrap around the protein element (Figure 2A). This region is free of crystallographic contacts in all structures reported here and its crystal structure likely reflects that in solution.

Figure 2.

Figure 2

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Structural basis for box C/D RNP specificity and the roles of the GAEK motif. The same coloring scheme as Figure 1 is used here. A. Structural features around the GAEK motif. Positions of GAEK (293-296) tetrapeptide are indicated by grey spheres. B. Schematic illustration of the contacts between Nop56/58 residues and the guide RNA in the halfmer RNP complex. Protein residues are indicated by colored oval and those in lighter color fall outside the GAEK motif. The asterisk indicates that the stacking interaction between F299 and U12 is observed in one of the protomers. C. Structure of the Δ33-34 RNP complex (-G33-C34) in the same orientation as that in A. The GAEK motif is completely disordered. D. Structure of the Δ1-9 RNP complex (-substrate) in the same orientation as that in A. Note that C11 and U12 are flipped towards α9A in the absence of the bound substrate.

The interaction between the GAEK motif and the guide RNA is non-specific for bases but highly shape complementary. The C-terminal extension interacts with the non-guide strand through both electrostatic and stacking interactions. Positively charged side chains are observed to interact with phosphate or to form cation-π stacking with nucleobases (Figure 2B). Unexpectedly, the GAEK tetrapeptide is not involved in direct interactions with either the guide or the substrate RNA. The fact that it immediately precedes helix α9A suggests its conserved role in the RNA-induced formation of α9A.

In order to assess the importance of the bound RNA to folding of the GEAK motif, we removed the last two nucleotide of the box C/D RNA (Δ33-34) that are tightly associated with α9A and obtained a 3.2 Å resolution crystal structure of the box C/D RNP containing Δ33-34 RNA and the substrate (-G33-C34, Figure 2C). Removal of the two guide RNA nucleotides completely destabilized α9A as indicated by the disappearance of its electronic density (Figure 2C), demonstrating the mutual induced fit principle in specific box C/D RNP assembly.

These analyses indicate that the GAEK motif-C/D RNA interaction occurs through mutually induced fit and demands an open-ended non canonical (NS) stem. This secondary structure feature distinguishes box C/D RNA from the homologous U4 snRNA whose NC stem is capped by a pentaloop. Correspondingly, the Nop56/58 homolog, hPrp31, does not have the GAEK-like motif (supplementary materials, Figure S1) and the similar peptide region is not involved in binding U4 snRNA (supplementary materials, Figure S2A & S2B). It is possible that this region of hPrp31 is involved in interacting with other RNAs.

Role of the GAEK motif in substrate placement

The α-helix α9A and its C-terminal extension containing the GAEK tetrapeptide that forms in the presence of the guide/substrate RNA creates a protein groove with L7Ae α2 (see secondary structure notation in Moore et al. (Moore et al., 2004) and Figure S1). This groove directs the emerging guide strand (Figure 1D) and its duplex with substrate RNA. As a result, the guide-substrate duplex is nearly perpendicular to the NS stem but parallel to the CS stem of the K-turn. Furthermore, the first two nucleotides of the substrate RNA (sG1, sA2) stack non-specifically on the C-terminal end of α9A that acts as a resting pole for the substrate RNA of even longer length (Figure 1D & 2A). The guide-substrate duplex follows the natural curvature of the surrounding protein surface that contacts the phosphate backbone of the substrate strand (Figure 1D). There are no specific interactions between the protein subunits and substrate nucleotides, thereby underlining the unique substrate binding mechanism in RNA-guided enzymes. Interestingly, the two guide-substrate duplexes in the asymmetric unit differ at position U12 of the guide RNA. In one of the duplexes, U12 is not paired with substrate nucleotide sA2 and instead is stabilized by stacking with Phe299 (Figure 2B), suggesting that this base pair is not required for correctly positioning the substrate RNA. Biochemical data have provided evidence for little or no substrate base pairing at this position but significant substrate base pairing at other positions (Appel and Maxwell, 2007).

The role of the substrate RNA itself and the GAEK motif interaction in directing the guide-substrate duplex is further demonstrated in two crystal structures that lack either one of the elements. In the previously described Δ33-34 complex, where the absence of G33 and C34 of the box C/D RNA led to destablization of α9A, a large portion of the guide-substrate duplex is disordered and the remaining portion follows a direction that is nearly perpendicular to that in the wild-type complex (Figure 2C). In addition, we crystallized a guide RNA lacking the guide region (Δ1-9) with L7Ae and a truncated Nop56/58 (amino acids 127-372, ΔNop56/58). The N-terminal truncation in ΔNop56/58 removed the fibrillarin binding domain but retained the RNA binding and the coiled-coil domains. In this complex, the first and second guide nucleotides, U12 and C11, point in the opposite direction to that in the substrate-bound complex (Figure 2D). Thus, the substrate RNA has the ability to further order and direct the guide RNA.

Substrate RNA-bound diRNP model predicts cross-RNP catalysis

In order to see if the previously determined diRNP structural model by electron microscopy (EM) (Bleichert et al., 2009) accommodates the substrate-bound halfmer RNP, we duplicated the halfmer RNP structure and manually docked it to the original RNP (Figure 3A). Remarkably, the two halfmer box C/D RNPs complement each other isologously at the R face without severe steric clashes. Furthermore, the core region (Nop56/58 without its N-terminal domain, L7Ae, and guide RNA) of the manually constructed diRNP fits reasonably well to the EM density of the diRNP as a rigid body (Figure 3B). The N-terminal domain of Nop56/58 and fibrillarin could not be docked into the density in their current conformations, reflecting their widely varied locations as previously noted (Aittaleb et al., 2003; Oruganti et al., 2007; Ye et al., 2009; Bleichert et al., 2009). Thus, the close agreement of the crystal structure-derived diRNP to the EM density of the substrate-free and fully assembled diRNP further validates the atomic model of diRNP with bound substrates.

Figure 3.

Figure 3

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The substrate-bound halfmer RNP structure is consistent with diRNP structure. A. Construction of the atomic diRNP model by fitting two identical crystal structures face-to-face without steric clashes. B. The core region of the manually constructed diRNP model (Nop56/58 is in green, L7Ae is in cyan, guide RNA is in red and substrate RNA is in yellow) was compared to the EM density map (clear). Single capital letters denote the protein subunits (F=fibrillarin, N=Nop56/58, L=L7Ae). Note that the guide-substrate duplexes are not in EM density, which highlights the functional difference between the two structural models. C. The substrate-bound diRNP in three different orientations. Note the similar projection dimensions as those of the electron microscopy structural model in B. Top, surface views; Bottom, ribbon diagrams;

In our diRNP model, the substrate RNA cannot interact with the fibrillarin associated with the same K-turn (Figure 4, cis fibrillarin) but can with the fibrillarin associated with the opposing K-turn (Figure 4, trans fibrillarin), especially if the fibrillarin is repositioned via a hinge motion to that previously observed in Af Nop56/58-fibrillarin structure (Figure 4, Af fibrillarin). At this position, the target nucleotide 2’-hydroxyl group is 10 Å from the methyltransfer donor S-adenosyl-L-methionine (SAM) bound to fibrillarin and can be positioned for catalysis with minor adjustments (Figure 4). Structural evidence for the described hinge motion in Nop56/58 already exists (Aittaleb et al., 2003; Oruganti et al., 2007; Ye et al., 2009; Bleichert et al., 2009). This analysis leads to an attractive hypothesis that diRNPs catalyse methylation reaction by a cross-RNP mechanism.

Figure 4.

Figure 4

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The substrate-bound diRNP model suggests cross-RNP catalysis. Structures of the modelled diRNP are shown on the left column and the corresponding cartoon representations are on the right column. A single guide-substrate RNA complex is shown for clarity. Fibrillarin is colored in blue, Nop56/58 is green and L7Ae is cyan. The guide RNA is in red and the substrate is in yellow. The target nucleotide is indicated by an orange sphere. Top shows the two fibrillarin positions of the diRNP and that of Af fibrillarin obtained by superimposing Af complex to the Pf complex. The fibrillarin associated with the same Nop56/58 bound to the guide RNA is labelled “cis fibrillarin” and the opposing fibrillarin is labelled “trans fibrillarin”. The previously determined Af Nop56/58-fibrillarin structure was superimposed to the opposing Pf RNP and the Af fibrillarin is labelled “Af fibrillarin”. Note that the “Af fibrillarin” is positioned to bind the guide-substrate duplex and the arrow indicates the predicted transition from the “trans” to “Af” position. Middle shows the same structure with fibrillarin molecules removed and the associated SAM molecules shown. SAM molecules are labelled as“trans SAM”, “Af SAM” and “cis SAM” respectively and is indicated by the red sphere in the cartoon representation. Note that “Af SAM” is the closest to the target methylation site and the arrow indicates the same transition as in top panel. Bottom panel shows the positional competition between Af fibrillarin and the bound L7Ae and suggests the release of L7Ae during substrate binding and methylation.

Mutational evidence for the cross-RNP catalysis

In order to test the importance of the structural elements critical for assembly and the hinge motion of fibrillarin, we constructed Pf Nop56/58 (Figure 5A) that either shortened the coiled-coil domain or lengthened the peptide hinge and examined the ability of the mutant Nop56/58 proteins in enzyme assembly and methylation. We shortened the coiled coil domain of Pf Nop56/58 (Nop56/58-Δ1cc) by removing one turn in each helix. We also inserted one (Nop56/58-In1) or three (Nop56/58-In3) amino acids to the Pf Nop56/58 peptide hinge region (Figure 5A).

Figure 5.

Figure 5

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Mutational data supporting cross-RNP catalysis and asymmetric assembly. A. Mutational scheme in cartoon representation. B. Crystal structure of the Pf Nop56/58-D1cc-fibrillarin complex (Nop56/58 is in green and fibrillarin is in blue) in comparison with that of the wild-type (purple). C. Gel mobility shift assay of RNP assembly with M. jannaschii sR8 RNA. Positions of shifted complexes are labelled on the left and proteins added are indicated on top. The lanes with Nop56/58-fibrillarin complexes all contain L7Ae. D. Methylation activities of the wild-type and mutant complexes. Activity is measured by saturated 3H counts on substrate RNA for various RNPs due to the transfer of methyl group from 3H-labeled S-adenosyl-L-methionion. “D” indicates methylation by box D guide and “D’” indicates methylation by box D’ guide.

A shortened coiled-coil is expected to change the longitudinal dimension of the Nop56/58 dimer and, therefore, orientation of the RNA binding surface. We confirmed this prediction by solving a crystal structure of Pf Nop56/58-Δ1cc-fibrillarin complex (Figure 5B). The mutant complex also forms a homodimer mediated by the coiled-coil in a similar manner as the wild-type complex but differs from the wild-type in its overall dimension and the relative orientation of Nop56/58 domains (Figure 5B). Notably, the coiled-coiled dimerization domain of the homologous U4 snRNP complex, hPrp31-15.5kD-U4, would be similar in length to that of Nop56/58-Δ1cc. Strikingly, while the Pf Nop56/58-Δ1cc was able to assemble into an RNP (Figure 5C), it completely abolished methylation activity of the enzyme (Figure 5D), suggesting a stringent requirement for correct orientation of protein domains in methylation.

The peptide hinge connecting the coiled-coil and the N-terminal domains was previously predicted to facilitate fibrillarin repositioning (Oruganti et al., 2007). Accordingly, neither Nop56/58-In1 nor Nop56/58-In3 affected assembly (Figure 5C). However, both mutations severely impaired the RNP’s ability in methylation, although Nop56/58-In1 maintained a substantial activity towards the substrate paired with the guide adjacent to box D (Figure 5D). These results are consistent with the occurrence of the hinge motion that most likely serves to reposition fibrillarin during dynamic methylation.

To provide further experimental support for that Af fibrillarin conformation is a functionally important state, we introduced a pair of cysteine residues in Af Nop56/58 (V73C/F288C) that are expected to lock the conformation of the N- and C-terminal domains, and therefore, that of fibrillarin (Figure 5A). We showed by the Ellman method (Creighton, 1989) that the prescribed disulfide bond is indeed formed (supplementary materials, Figure S3) and tested the ability of the cross-linked Af Nop56/58 in RNP assembly and catalysis. Consistent with the structural prediction, the Af V73C/F288C-fibrillarin complex was fully active in RNP assembly and catalysis and to some extent, had higher activity than the wild-type (Figure 5C & Figure 5D). This result further supports the model of dynamic methylation.

Substrate-bound diRNP model predicts an asymmetric holoenzyme

Earlier biochemical studies suggested the possibility that asymmetrically assembled diRNPs may represent an important functional state. Results of the in vivo chemical cross-linking and nucleotide analog interference mapping experiments show that the 15.5kD protein is associated with the terminal box C/D module and fibrillarin interacts more extensively with the internal box C/D module (box C’/D’) where 15.5kD is absent (Cahill et al., 2002; Szewczak et al., 2002). Furthermore, in vitro binding studies showed that archaeal box C/D RNPs are often symmetric with respect to L7Ae binding but can be asymmetric with certain internal loop sequences (Gagnon et al., 2006). Our substrate-bound diRNP model provides some support for these important experimental observations.

In order for fibrillarin to reach the target nucleotide, it is necessary for it to be positioned as that in the Af Nop56/58-fibrillarin complex. This position is supported by the activity of the cross-linked Af Nop56/58-fibrillarin complex described above. This position, however, is also where L7Ae binds in a fully assembled RNP (Figure 4, bottom). We thus predict that L7Ae is absent from the site of catalysis where the RNA is primarily anchored through the interaction between the guide-substrate duplex and fibrillarin. In addition to accommodate the catalytic subunit, the release of L7Ae also facilitates substrate RNA binding to diRNP. As predicted by Bleichert et al. and suggested by our diRNP model, box C/D RNA do not follow the direction of the coiled-coil domain. Rather, they bind across two RNPs (Figure 6A). This mode of binding is consistent with the optimal separation of 10-12 bases between two box C/D modules (Tran et al., 2005). Our substrate-bound halfmer RNP structure further shows that it is difficult for the two K-turns associated with substrates to make a smooth connection across two halfmer RNPs due to their different paths (Figure 6B & 6C). This dilemma could be resolved by the asymmetric model discussed above in which the site lacking L7Ae releases the K-turn in order for the substrate RNA to bind to diRNP (Figure 6D).

Figure 6.

Figure 6

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Illustration of four possible guide RNA paths (red) in the diRNP model. Panels are of the same view as the first in Figure 4B and ± L7Ae indicates the presence and absence of L7Ae at each K-turn. A. Substrate-free guide RNA (from the Δ1-9 structure) binds across RNP with sufficient flexibility in the internal loop region and with L7Ae bound (+L7Ae). B. & C. Doubly or singly bound substrate RNA interrupts connectivity between two K-turns that are bound with L7Ae. D. Singly bound substrate to each guide RNA (from the full complex structure) accommodates cross-RNP binding model if one K-turn is free of L7Ae (-L7Ae) that allows a slight rotation of the guide RNA.

The predicted asymmetric methylation model is already found support in previous studies of eukaryotic and archaeal box C/D RNP assembly. The eukaryotic homolog of L7Ae, the 15.5kD protein, was found to be asymmetrically associated with the box C/D and box C’/D’ motifs (Cahill et al., 2002; Szewczak et al., 2002). In a more recent experiment with a mutant Methanococcus jannaschii L7Ae (L7Ae EL9-K26Q) that selectively binds box C/D motif only, an asymmetrically assembled archaeal box C/D RNP was constructed (Gagnon et al., 2010). Strikingly, the asymmetrically assembled box C/D RNP catalyzed methylation of the substrate paired with either D or D’ guide more efficiently than the wild-type enzyme (Gagnon et al., 2010). Paradoxically, the asymmetrically assembled box C/D RNP was found to contain at least two copies of the single box C/D motif binding L7Ae EL9-K26Q protein. This result could be explained by a weak but direct association between L7Ae EL9-K26Q and Nop56/58 in the classical bipartite assembly model. However, no direct interaction between L7Ae and Nop56/58 has been observed. Thus, this result is better explained by the diRNP model without invoking a direct L7Ae-Nop56/58 interaction. Taken together, simultaneous binding of L7Ae (or 15.5kD) to both box C/D and C’/D’ motifs is not necessary and, may in fact, be inhibitory to efficient methylation as predicted by the model of asymmetric methylation model.

Discussion

This work provides insights into two important aspects of the methylation RNP function. First, the high resolution halfmer RNP structures delineate a remarkable molecular mechanism for specific assembly that distinguishes the methylation RNP from the homologous U4 snRNP. Second, the substrate-bound diRNP model suggests a surprising cross-RNP catalysis strategy and predicts an asymmetric holoenzyme.

Box C/D RNP assembles differently than U4 snRNP

Previously, interesting comparisons have been drawn between the eukaryotic box C/D RNP and the spliceosomal 15.5 kD-U4 snRNA complex. In mammals, 15.5kD protein helps to form box C/D RNPs and also assemble the spliceosome by binding to U4 snRNA (Nottrott et al., 2002). The 15.5 kD-U4 complex facilitates subsequent binding of the splicing factor, Prp31(Nottrott et al., 2002). Prp31 shares a strong homology in its RNA binding domain (or Nop domain) with the Nop56/58 protein and is believed to bind the spliceosomal 15.5kD-U4 complex in a manner similar to the way Nop56/58 binds the 15.5 kD-bound box C/D RNA. Structural and functional studies of human spliceosomal protein hPrp31 have shed light on how its Nop domain interacts with the 15.5 kD-U4 stem II complex (Liu et al., 2007) but could not explain the specificity of Nop56 or Nop58 for the 15.5kD- box C/D RNA complex. Despite the high degree of homology between hPrp31 and Nop56/58, the Nop domain of Nop56/58 interacts specifically with the composite surface formed by both box C/D RNA and L7Ae and the guide RNA fork. The Nop domain of hPrp31 interacts, in addition to a composite surface formed by U4 RNA and 15.5kD protein, with the U4-specific pentaloop (Liu et al., 2007). This result explains the previous biochemical data on RNP assembly and provides a structural basis for distinct RNP assembly.

A cross-RNP model of assembly and catalysis

Our data is consistent with an assembly model based on the diRNP structure visualized by electron microscopy (Bleichert et al., 2009). Our data further predicts a cross-RNP catalysis mechanism based on the position of the bound substrate RNA. The guide and substrate RNAs are strategically oriented by the well conserved GAEK motif. This mode of binding is likely maintained in the full diRNP structure. The substrate-bound diRNP model shows that the target nucleotide is stably tethered by Nop56/58, and to some extent by L7Ae, to be contacted and modified by the fibrillarin associated with the opposing Nop56/58 in the other halfmer RNP. The fact that the experimentally observed Af fibrillarin can contact the target nucleotide further predicts the functional state of the catalytic subunit, thereby increasing the confidence in the cross-RNP catalysis model.

In the traditional bipartite assembly model where the box C/D RNA binds along a single RNP, the target nucleotide would be modified by the fibrillarin associated with the same Nop56/58 that also holds the substrate RNA. In this model, the protein surface surrounding the target nucleotide makes it difficult for the fibrillarin to contact it, even if the fibrillarin repositions. The cross-RNP strategy suggested by the diRNP model resolves this problem by supplying the catalytic subunit in trans. The substrate-bound diRNP model, therefore, provides a valuable perspective in understanding box C/D RNP function.

The observed symmetric archaeal apoenzyme and the predicted asymmetric holoenzyme raise an interesting mechanistic question, namely, does box C/D RNP dynamically change its conformation upon substrate binding or does it maintain a distribution of static conformations? Evidence is available for both archaeal and human box C/D RNPs that suggests a strong likelihood of conformational changes in box C/D RNP function. Human box C/D RNP assembly requires multiple biogenesis factors including ATPases (McKeegan et al., 2007; McKeegan et al., 2009). Temperature plays a key role in symmetric assembly of archaeal box C/D RNPs (Gagnon et al., 2006). However, experiments focusing on substrate-induced conformational changes have not been carried out. Thus, determination of the exact mechanism of the predicted symmetric to asymmetric transition requires further studies.

The model of asymmetric holoenzyme provides an opportunity to understand how box C/D RNPs capture large and complex substrate RNAs. In a symmetrically assembled box C/D RNP, the internal loop possessing two guides is closed by the bound L7Ae at both ends (Figure 7). In the asymmetric model, however, one end of the internal loop is freed, which significantly enlarges the internal loop opening (Figure 7). The rRNA or snRNA substrates must make 10-21 base pairs with one strand of the internal loop. It is clearly more difficult for rRNA or snRNA to thread through a small opening internal loop (linking number ΔL = +1) than to loop through a large opening internal loop (linking number ΔL=0) (Figure 7). We suggest that both formation of the RNA fork by the GAEK motif and the asymmetric distribution of L7Ae/15.5kD protein on box C/D RNA, therefore, provide a necessary means for the substrate-guide interaction to take place (Figure 7).

Figure 7.

Figure 7

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Schematics of the observed symmetric substrate-free diRNP structure and the proposed asymmetric holoenzyme. Possible substrate interactions with the symmetric and asymmetric assemblies are illustrated by yellow substrate RNA.

Experimental Procedures

Protein purification, crystallization and structure determination

The gene encoding recombinant Pf Nop56/58 or the N-terminal domain truncated ΔNop56/58, and Pf L7Ae and an N-terminal 6xHistidine tag were cloned into a pET28a vector. The gene encoding recombinant Pf fibrillarin was cloned into a pET24a vector. The proteins were over-expressed in Escherichia coli BL21(DE3) and purified from a Ni-NTA column followed by Superdex 200 gel filtration (Amersham Pharmacia) as previously described (Oruganti et al., 2007). All synthetic RNA oligos were ordered from Integrated DNA Technologies and purified according to manufacture instructions. The complexes were similarly formed at a 1.2:1.2:1:1 (1.2:1:1) molar ratio of RNA:L7Ae:Nop56/58:fibrillarin (RNA:L7Ae:ΔNop56/58) with a total concentration of 46-108 mg ml−1 before crystallization. Crystals of the three RNPs were formed at 30°C in hanging drops. The full complex was equilibrated with reservoir solutions containing 0-400 mM potassium chloride, 200 mM-1.5 M sodium chloride, 150-250 mM magnesium acetate, 200 mM ammonium acetate, 50 mM HEPES-NaOH (pH 7.0), and 0–5% PEG 4000. The Δ1-9 complex was equilibrated with reservoir solutions containing 200 mM potassium chloride, 5 mM magnesium chloride, 50 mM TrisHCl (pH 7.5), and 4%–8% PEG4000 or PEG3350. The Δ33-34 complex used reservoir solutions containing 1.8 M potassium chloride, 40 mM magnesium chloride, 50 mM MES pH 5.6, and 3% PEG8000. All crystals were soaked stepwise in cryosolutions containing the mother liquor plus 10% (v/v) and 15% (v/v) glycerol, respectively, before being flash cooled in a liquid nitrogen stream for data collection. Diffraction data sets were collected at the South Eastern Consortium Access Team (SER-CAT) beamline 22ID and was processed using HKL2000 (Otwinowski and Minor, 1997) (Table S1). All three complex crystals contain two RNPs in the asymmetric unit and have estimated solvent contents of 63%-68%. The full complex crystals are in C2 space group with cell dimensions a = 293.5 Å, b = 94.1 Å, c = 96.8 Å, and β 101.47°. The Δ1-9 complex crystals are in P212121 space group with cell dimensions a = 87.2 Å, b = 91.8 Å, and c =155.6 Å. The Δ33-34 complex crystals are in P21212 space group with cell dimensions a = 330.0 Å, b = 94.6 Å, and c = 97.7 Å. The three structures were determined by a molecular replacement method (MOLREP) (Vagin and Teplyakov, 1997) using either ΔNop56/58 or full-length Nop56/58-fibrillarin monomeric complex (Oruganti et al., 2007) as the search model. In all cases, two solutions were found that correctly reproduced the typical dimer observed in isolated Nop56/58 complex structures. Electron densities phased with proteins alone clearly indicated location of L7Ae and RNA and allowed their placement in the complex. The RNP structures were refined to satisfactory statistics (Table S1) using CNS (Brunger et al., 1998), REFMAC5 (Murshudov et al., 1997) or PHENIX (Adams et al., 2002). The following programs were employed for modelling and displaying: COOT (Emsley and Cowtan, 2004), O (Jones et al., 1991), and Pymol (DeLano).

To generate the duplicated halfmer RNP, transformation with Euler angles (°) (55.3, 163.2, 27.8) and translation vector (Å) = (33.9 6.8 9.4) was applied to the original halfmer RNP. The duplicated halfmer RNP was then fit by hand to the original halfmer RNP to generate the diRNP guided by the overall shape of the EM model. To fit the core region of the manually constructed diRNP model to EM density, we first removed the N-terminal domain of Nop56/58, fibrillarin, and the bound RNA from the manually constructed diRNP coordinates. We then fitted the remaining core protein complex as a rigid body to the EM density (EMD-1636) manually and by the “fit in map” procedure in program Chimera (http://www.cgl.ucsf.edu/chimera). The bound guide and substrate RNA molecules were later added to the fitted complex. No effort was made to fit the N-terminal domain of Nop56/58 and fibrillarin, which was explored in details by Bleichert et al. (Bleichert et al., 2009).

Crystals of the Pf Nop56/58-Δ1cc-fibrillarin complex were obtained similarly as those of the wild-type Pf Nop56/58-fibrillarin complex (Oruganti et al., 2007). The crystals are in the same space group as those of the wild-type complex but have a short c-axis of the unit cell (a=100.2 Å, b=100.2Å, c=265.4Å versus a=100.8Å, b=100.8Å, c=283.2Å). The structure was determined by the molecular replacement method and refined to satisfactory statistics using PHENIX (Adams et al., 2002).

RNA assembly, in vitro methylation, and EMSA analysis

Plasmids carrying the genes encoding Pf Nop56/58 and Af Nop56/58 mutants were obtained using Quikchange kits and the mutant proteins were purified similarly as the wild-type proteins. RNP complexes were assembled and methyltransferase activity assessed as previously described (Tran et al., 2003; Zhang et al., 2006). Briefly, RNP complexes were assembled at 70°C for 10 mins in 80 ul reaction volumes. Standard assembly reactions were 0.65 uM of 5’ radiolabeled sR8 box C/D sRNA (~ 1×104 cpm), 0.8 uM L7Ae, 3.1 μM Nop56/58, and 1.1 μM fibrillarin. Assembled RNPs were then incubated with 6.5 μM target RNA (IDT), 3.3 μM S-adenosyl-L-methionion S-adenosyl-L-methionion (SAM) (Calbiochem), and 3 μl of 3H-SAM (55 mCi/ml; MP Biomedicals) in a final volume of 110 μl. After incubation at 68°C for various times (10-60 minutes), 20 μl aliquots were removed and TCA precipitated onto filter paper. Methyl incorporation into target RNAs was measured by scintillation counting. Methylation assays were performed in triplicate and reported as the average of three experiments. Electrophoretic mobility-shift analysis of assembled RNPs was carried out on native 4-6% polyacrylamide gels containing 2% glycerol and buffered with 25 mM potassium phosphate (pH 7.0). Polyacrylamide gels were dried and visualized by autoradiography or phosphorimaging.

Supplementary Material

01

Table 1.

Data collection and refinement statistics (values in parentheses refer to those of the highest resolution shell)

full complex (PDBid: 3NMU) Δ1-9 complex* (PDBid: 3NVI) Δ33-34 complex* (PDBid: 3NVK) Δ1cc complex* (PDBid: 3NVM)
diffraction data
 Space group C2 P212121 P21212 P3112
 Unit-cell parameters (Å)
 a 293.5 87.2 330 100.6
 b 94.1 91.8 94.548 100.6
 c 96.8 155.6 97.729 265.4
 Resolution range (Å) 50.0-2.7 (2.8-2.7) 50.0-2.7 (2.8-2.7) 40.0-3.20 (3.3-3.2) 50-3.4 (3.5-3.4)
 No. of unique reflections 61447 (3987) 32071 (2671) 42641 (3238) 32901(1098)
 Redundancy 5.8 (4.5) 6.9 (4.1) 8.8 (4.1) 5.2(2.6)
 Completeness (%) 89.2 (58.1) 99.2 (92.1) 83.4 (64.3) 80.0(26.8)
 I/σ(I) 26.2 (2.2) 32.1 (2.0) 21.1 (2.2) 20.1(1.5)
 Rsym (%) 7.4 (51.4) 9.6 (61.8) 10.7 (53.3) 9.3(44.5)
Refinement statistics
 Resolution Range (Å) 50.0-2.7 (2.8-2.7) 50.0-2.7 (2.8-2.7) 40.0-3.2 (3.3-3.2) 50.0-3.4(3.5-3.4)
 Rwork (%) 22.0 (32.8) 21.5 (31.9) 23.1 (32.8) 26.5 (41.1)
 Rfree (%) 28.6 (39.2) 26.4 (37.0) 28.6 (35.2) 32.3 (43.2)
 Model information
 No. of protein-RNA complexes 2 2 2 1
 No. of amino-acid/nucleotide/ligand 1426/93/2 734/48/0 1420/54/2 575/0/0
 No. of protein/RNA atoms 11440/1994/52 5814/1026 11374/1162/52 4598/0/0
 R.m.s. deviations of the model
 Bond length (Å) 0.09 0.007 0.009 0.012
 Bond angle (°) 1.35 1.21 1.523 1.791
 Average B-factors
 Nop56/58:L7Ae :fibrillarin:RNA 82.8:120:73.2:170.5 95.6:144.7:82.1:193.4 86.4:100.7:92.8 161.0:154.1
Ramachandran plot
Residues in most favored region 1169 [81.9%] 654 [89.0%] 1046[73.6%] 414[72.1%]
Residues in additionally allowed region 185 [13.0%] 74 [10.2%] 330 [23.3%] 121[21.2%]
Residues in generously allowed region 72 [5%] 6 [0.08%] 44 [3.1%] 40[6.7%]
Residues in disallowed region 0 [0%] 0 [0%] 0 [0%] 0 [0%]
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*

Δ1–9 complex refers to Pf Nop56/58-fibrillarin complex bound to L7Ae and box C/D RNA 10-33 without the substrate RNA; Δ33-34 complex refers to Pf Nop56/58-fibrillarin complex bound to L7Ae and box C/D RNA 1-32 with the substrate RNA; Δ1cc complex refers to Pf Nop56/58- Δ1cc mutant (deletion of one turn of coiled-coil) bound to fibrillarin without L7Ae or RNA.

Acknowledgments

This work was supported in part by National Institutes of Health grants R01GM066958 (H.L) and R01GM54682 (MT and RT) and NSF grant MCB 0543741 (ESM). S. Xue is supported by an American Heart Association Florida/Puerto Rico Affiliate postdoctoral fellow (0725583B). X-ray diffraction data were collected the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID and 22-BM beamlines at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions for APS beamlines may be found at http://necat.chem.cornell.edu/ and www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38.

Footnotes

DATA BASE ACCESSION NUMBERS:

Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 3NMU (full complex), 3NVI (Δ1-9 complex), 3NVK (Δ33-34 complex) and 3NVM (Δ1cc complex).

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