2.1 Core components of the box C/D RNP

Box C/D RNAs were first detected by nuclear fractionation as a class of RNAs specifically localized to the nucleolus (Reddy et al. Reference Reddy, Li, Henning, Choi, Nohga and Busch1981) and immunoprecipitated by the nucleolar protein fibrillarin (Tyc & Steitz, Reference Tyc and Steitz1989). Box C/D RNAs contain two sets of conserved sequence motifs, box C or C′ (RUGAUGA, where R is any purine) and D or D′ (CUGA). In addition, a conserved stem is formed between the 5′ and 3′ ends of the box C/D RNA that forms a hairpin-like structure (Fig. 2). The internal loop flanked by the box C/D and box C′/D′ motifs is complementary to the sequence of substrate RNA and thus serves as the substrate specificity element. Remarkably, methylation takes place via a ruler mechanism in which the substrate nucleotide that is paired with the fifth nucleotide upstream of box D or D′ is modified. In archaea, an optimal length of the internal loop is found to be 10–12 nucleotides (Tran et al. Reference Tran, Zhang, Lackey and Maxwell2005).

Fig. 2.

Conserved features of RNP modification enzymes. (a) Box C/D MTases comprise box C/D RNA and four core proteins. (b) Box H/ACA pseudouridylases comprise box H/ACA RNA and four core proteins. (c) hTR is a special member of the box H/ACA RNPs without known modification activities. The table lists names of the core proteins associated with each type of RNPs in both eukarya and archaea.

Following the identification of fibrillarin as the box C/D RNA-associated protein (Tyc & Steitz, Reference Tyc and Steitz1989), genetic synthetic lethal screens with a fibrillarin mutant revealed two additional proteins, Nop56p and Nop58p, that are associated with box C/D RNA (Gautier et al. Reference Gautier, Berges, Tollervey and Hurt1997; Lafontaine & Tollervey, Reference Lafontaine and Tollervey1999, Reference Lafontaine and Tollervey2000; Wu et al. Reference Wu, Brockenbrough, Metcalfe, Chen and Aris1998). Finally, tag-purification of U3-snoRNP followed by mass spectrometry identification revealed the fourth box C/D RNP protein, Snu13p (15·5 K in mammals and L7Ae in archaea) (Watkins et al. Reference Watkins, Segault, Charpentier, Nottrott, Fabrizio, Bachi, Wilm, Rosbash, Branlant and Luhrmann2000). Fibrillarin, which contains an SAM binding domain, is the subunit responsible for the methyltransfer reaction. Nop56p and Nop58p are homologous to each other and to a single Nop56/58 protein in archaea (Lafontaine & Tollervey, Reference Lafontaine and Tollervey2000; Omer et al. Reference Omer, Lowe, Russell, Ebhardt, Eddy and Dennis2000). These four proteins are found in all members of the box C/D RNPs including those required for rRNA cleavage. Notably, yeast U3 snoRNPs in isolated small ribosome processing complexes contain more than 20 additional proteins that are most likely required for U3-specific functions (Dragon et al. Reference Dragon, Gallagher, Compagnone-Post, Mitchell, Porwancher, Wehner, Wormsley, Settlage, Shabanowitz, Osheim, Beyer, Hunt and Baserga2002; Grandi et al. Reference Grandi, Rybin, Bassler, Petfalski, Strauss, Marzioch, Schafer, Kuster, Tschochner, Tollervey, Gavin and Hurt2002; Krogan et al. Reference Krogan, Peng, Cagney, Robinson, Haw, Zhong, Guo, Zhang, Canadien, Richards, Beattie, Lalev, Zhang, Davierwala, Mnaimneh, Starostine, Tikuisis, Grigull, Datta, Bray, Hughes, Emili and Greenblatt2004; Perez-Fernandez et al. Reference Perez-Fernandez, Roman, de Las Rivas, Bustelo and Dosil2007).

2.2 Core components of the box H/ACA RNPs

Box H/ACA RNAs were also discovered through the isolation of nucleolus-specific RNAs (Kiss & Filipowicz, Reference Kiss and Filipowicz1993), but were immunoprecipitated by another nucleolus protein, Gar1 (Girard et al. Reference Girard, Lehtonen, Caizergues-Ferrer, Amalric, Tollervey and Lapeyre1992). The box H/ACA sno/scaRNAs contains a central unit of the hairpin–hinge–hairpin–tail secondary structure as shown in Fig. 2. In archaea, box H/ACA RNAs can contain one, two, or three hairpin structures, each with an internal loop – the pseudouridine (Ψ) pocket – complementary to a substrate RNA (Rozhdestvensky et al. Reference Rozhdestvensky, Tang, Tchirkova, Brosius, Bachellerie and Huttenhofer2003; Tang et al. Reference Tang, Bachellerie, Rozhdestvensky, Bortolin, Huber, Drungowski, Elge, Brosius and Huttenhofer2002). Box H refers to the conserved sequence (ANANNA, N means any nucleotide) in the hinge connecting two hairpin units and box ACA is the conserved trinucleotide sequence at the 3′ tail of the RNA. The uridine targeted for modification is positioned at the center of the Ψ pocket and is typically 14–16 nucleotides away from box H or box ACA.

The first H/ACA RNP protein identified is yeast Gar1p, from a screen for proteins containing glycine-arginine-rich domains similar to that of fibrillarin (Girard et al. Reference Girard, Lehtonen, Caizergues-Ferrer, Amalric, Tollervey and Lapeyre1992). Purification of Gar1p-bound RNPs revealed H/ACA RNAs and three additional proteins associated with H/ACA RNAs (Henras et al. Reference Henras, Henry, Bousquet-Antonelli, Noaillac-Depeyre, Gelugne and Caizergues-Ferrer1998; Lafontaine et al. Reference Lafontaine, Bousquet-Antonelli, Henry, Michèle Caizergues and Tollervey1998; Watkins et al. Reference Watkins, Gottschalk, Neubauer, Kastner, Fabrizio, Mann and Luhrmann1998). In yeast, these correspond to Cbf5p (Dyskerin in human, NAP57 in mice, Cbf5 in archaea), Nhp2p (NHP2 in mammals, L7Ae in archaea) and Nop10p (Fig. 2). Cbf5p contains two of the three hallmark sequence motifs of pseudouridine synthases (motifs I and II) including the strictly conserved asparate residue in motif II (Koonin, Reference Koonin1996) and is thus the putative catalytic subunit. Nhp2p shares sequence similarity with the box C/D core protein, 15·5 kDa/Snu13p/L7Ae, but lacks K-turn binding specificity. This homology suggests that the two classes of sno/scaRNPs share a common origin. All four proteins are also components of the human telomerase (hTR) and are required for the biogenesis of hTR (Collins, Reference Collins2006).

Mutations of the protein components of sno/scaRNPs have been implicated in multiple human disorders. However, it has not been firmly established whether these mutations cause defects in the modification function itself or other functions requiring the sno/scaRNP proteins. Fibrillarin, the enzymatic component of box C/D RNPs, is essential for development and its depletion is embryonic lethal (Newton et al. Reference Newton, Petfalski, Tollervey and Caceres2003). Mutations in the human dyskerin gene DKC1 (Cbf5) (Heiss et al. Reference Heiss, Knight, Vulliamy, Klauck, Wiemann, Mason, Poustka and Dokal1998; Marrone & Mason, Reference Marrone and Mason2003; Meier, Reference Meier2005), NHP2 (Vulliamy et al. Reference Vulliamy, Beswick, Kirwan, Marrone, Digweed, Walne and Dokal2008) and NOP10 gene (Vulliamy et al. Reference Vulliamy, Beswick, Kirwan, Marrone, Digweed, Walne and Dokal2008; Walne & Dokal, Reference Walne and Dokal2008) are associated with the X-linked genetic disorder, dyskeratosis congenita (DC). This genetic disease is characterized by cutaneous pigmentation, bone marrow failure, nail dystrophy, white potentially malignant patches on the oral mucosa, continuous tear production and often abnormally low blood platelet counts, anemia and predispositions to epithelial cancers (Marrone & Mason, Reference Marrone and Mason2003). Importantly, the box H/ACA core proteins are also essential to hTR biogenesis (Collins, Reference Collins2006; Mitchell et al. Reference Mitchell, Wood and Collins1999b). They are associated with the 3′-end of the telomerase RNA and are required for the accumulation, 3′-end processing and nuclear localization of hTR in Cajal bodies. In DC patients, malfunction of rRNA and shortening of telomeres have been observed (Marrone et al. Reference Marrone, Walne and Dokal2005; Mochizuki et al. Reference Mochizuki, He, Kulkarni, Bessler and Mason2004).

4.1 Maturation of the sno/scaRNAs

The C/D and H/ACA RNAs are maturated through elaborative pathways that generally include transcription, 5′ and 3′ processing and localization. This process requires a number of biogenesis factors and depends on other RNA functional processes (Kiss et al. Reference Kiss, Fayet, Jady, Richard and Weber2006; Matera et al. Reference Matera, Terns and Terns2007).

The majority of mammalian box C/D and box H/ACA sno/scaRNAs responsible for guiding modifications are transcribed by RNA polymerase II (Pol II) as pre-mRNA introns (Fig. 3). Therefore, mRNA splicing also influences the generation of linear sno/scaRNAs precursors. Most box C/D RNAs are located 65–85 nucleotides upstream of the 3′ splice site and ~50 nucleotides upstream of the branch site (Hirose & Steitz, Reference Hirose and Steitz2001), whereas H/ACA RNAs have no preferential intronic location relative to the 5′ or 3′ splice sites of the host intron (Richard et al. Reference Richard, Kiss, Darzacq and Kiss2006; Schattner et al. Reference Schattner, Barberan-Soler and Lowe2006). Correspondingly, the processing of box C/D RNAs but not box H/ACA RNAs depends on general splicing factors (Hirose et al. Reference Hirose, Ideue, Nagai, Hagiwara, Shu and Steitz2006). The intronic sno/scaRNAs undergo debranching and exonucleolytic trimming of both 5′- and 3′-ends (Allmang et al. Reference Allmang, Kufel, Chanfreau, Mitchell, Petfalski and Tollervey1999). In lower eukaryotes, such as yeast and plants, sno/scaRNAs are processed from both non-intronic and splicing-independent intronic transcripts (Chanfreau et al. Reference Chanfreau, Rotondo, Legrain and Jacquier1998; Leader et al. Reference Leader, Clark, Watters, Beven, Shaw and Brown1999; Qu et al. Reference Qu, Henras, Lu, Zhou, Zhou, Zhu, Zhao, Henry, Caizergues-Ferrer and Bachellerie1999).

Processed box C/D and box H/ACA RNAs are associated with either all or most RNP core proteins and are translocated into Cajal bodies before the mature snoRNPs relocate to nucleolus (Fig. 3). Factors or processes that facilitate the orderly localization events are yet to be completely characterized. Transport of human U3 snoRNA was shown to be facilitated by the snRNA export adaptor, PHAX, and the export receptor, CRM1 (Boulon et al. Reference Boulon, Verheggen, Jady, Girard, Pescia, Paul, Ospina, Kiss, Matera, Bordonne and Bertrand2004). On the other hand, a conserved sequence box (CAB box) is responsible for the retention of scaRNAs in Cajal bodies through its specific interaction with a WD40 protein, WDR79. In general, the WD40 family of proteins has a circularized beta-propeller structure and forms platforms for the assembly of protein complexes associated with a wide range of biological pathways. WDR79 has been found to interact directly with the CAB box of both box C/D and box H/ACA scaRNAs, as well as telomerase RNA in Cajal bodies (Jady et al. Reference Jady, Bertrand and Kiss2004; Tycowski et al. Reference Tycowski, Shu, Kukoyi and Steitz2009; Venteicher et al. Reference Venteicher, Abreu, Meng, Mccann, Terns, Veenstra, Terns and Artandi2009).

4.2 Assembly of sno/scaRNPs

The processed sno/scaRNAs are assembled with core proteins at the stage of pre-mRNA splicing or co-transcriptionally in order to avoid the degradation pathway that is known to target non-functional introns. A number of trans-acting factors have been identified that affect the assembly of mammalian sno/scaRNPs (Boulon et al. Reference Boulon, Marmier-Gourrier, Pradet-Balade, Wurth, Verheggen, Jady, Rothe, Pescia, Robert, Kiss, Bardoni, Krol, Branlant, Allmang, Bertrand and Charpentier2008; King et al. Reference King, Decatur, Bertrand, Maxwell and Fournier2001; Newman et al. Reference Newman, Kuhn, Shanab and Maxwell2000; Venteicher et al. Reference Venteicher, Meng, Mason, Veenstra and Artandi2008), suggesting the existence of conformational barriers to achieve assembly. This property may explain why in vitro reconstituted eukaryotic sno/scaRNPs have no enzymatic activities (Maxwell, E. S. and Meier, U. T., personal communications) and the archaeal particles require high temperature to activate assembly and enzyme activities (Baker et al. Reference Baker, Youssef, Chastkofsky, Dy, Terns and Terns2005; Charpentier et al. Reference Charpentier, Muller and Branlant2005; Gagnon et al. Reference Gagnon, Zhang, Agris and Maxwell2006; Omer et al. Reference Omer, Ziesche, Ebhardt and Dennis2002).

Box C/D sno/scaRNPs assembly begins at the C1 splicing complex stage at which the first transesterification reaction takes place with the recruitment of the 15·5 K protein (Hirose et al. Reference Hirose, Shu and Steitz2003). The 15·5 K/NHPX protein interacts with box C/D RNA in a splicing-dependent manner (Hirose et al. Reference Hirose, Shu and Steitz2003). In addition, in vitro studies with its archaeal homolog, L7Ae, identified this protein as the initiator of box C/D RNP assembly (Rashid et al. Reference Rashid, Aittaleb, Chen, Spiegel, Demeler and Li2003; Tran et al. Reference Tran, Zhang and Maxwell2003). Thus, box C/D RNP assembly likely begins, while the spliceosome is still assembled around the pre-mRNA. In vivo chemical cross-linking experiments place 15·5 K/NHPX, fibrillarin and Nop58 close to the terminal box C/D motif and fibrillarin and Nop56 to the box C′/D′ motif (Cahill et al. Reference Cahill, Friend, Speckmann, Li, Terns, Terns and Steitz2002).

In vivo assembly of box H/ACA sno/scaRNPs is found to begin during transcription elongation (Richard et al. Reference Richard, Kiss, Darzacq and Kiss2006) and depends on several conserved trans-acting factors (Grozdanov et al. Reference Grozdanov, Roy, Kittur and Meier2009; Kittur et al. Reference Kittur, Darzacq, Roy, Singer and Meier2006; Yang et al. Reference Yang, Rotondo, Porras, Legrain and Chanfreau2002, Reference Yang, Hoareau, Froment, Monsarrat, Henry and Chanfreau2005b). The most extensively characterized factors include Naf1 and Shq1. Naf1, a protein specifically bound to the C-terminal domain (CTD) of Pol II, is associated with newly transcribed box H/ACA RNAs and dyskerin/Cbf5. Naf1 contains a domain structurally homologous to Gar1 and is believed to compete with Gar1 for binding to dyskerin/Cbf5 (Darzacq et al. Reference Darzacq, Kittur, Roy, Shav-Tal, Singer and Meier2006; Leulliot et al. Reference Leulliot, Godin, Hoareau-Aveilla, Quevillon-Cheruel, Varani, Henry and Van Tilbeurgh2007). Shq1 is another factor that is required for the accumulation of box H/ACA RNAs and forms a complex with Naf1 (Yang et al. Reference Yang, Rotondo, Porras, Legrain and Chanfreau2002). The CTD of Shq1 is also shown to bind Cbf5 and have stand-alone chaperone activity in vitro (Godin et al. Reference Godin, Walbott, Leulliot, van Tilbeurgh and Varani2009). In addition, general chaperones and nucleic acid-associated ATPases are found to facilitate box H/ACA RNP assembly (Kiss et al. Reference Kiss, Fayet-Lebaron and Jady2010).

5.1 Methyltransferases and fibrillarin

Methyltransferases (MTases) are found as proteins that function independently (stand-alone) or as ribonucleoprotein particles that rely upon RNA. The RNA-guided MTases have thus far been found to strictly target RNA ribose modification, while stand-alone enzymes target a much wider range of substrates and functional groups including proteins, DNA, RNA (ribose and base constituents), lipids and small molecules for methylation. In general, MTases can be SAM-dependent and SAM-independent. From primary structural features, and regardless of need for an RNA guide, the SAM-dependent MTases are divided into five classes (Class I–V MTases) (Schubert et al. Reference Schubert, Blumenthal and Cheng2003), with most of the 2′-O-MTases belonging to Class I and Class IV. The Class I MTases share little sequence identity, but have a common structural domain comprising a central seven β-sheet mixed with α-helices (Fig. 4a) (Cheng & Roberts, Reference Cheng and Roberts2001; Martin & McMillan, Reference Martin and Mcmillan2002). The Class IV MTases, also known as SPOUT (SpoU-TrmD) superfamily MTases, exhibit a Rossmann α/β-fold with a deep trefoil knot (Anantharaman et al. Reference Anantharaman, Koonin and Aravind2002; Nureki et al. Reference Nureki, Shirouzu, Hashimoto, Ishitani, Terada, Tamakoshi, Oshima, Chijimatsu, Takio, Vassylyev, Shibata, Inoue, Kuramitsu and Yokoyama2002) (Fig. 4b). The difference in the central domain fold reflects the different substrate specificity. Fibrillarin, the MTase in sno/scaRNPs, was found to have structural homologies to Class I bacterial SAM-dependent MTases. These include FtsJ (PDB code: 1EJ0) and AviRa (PDB code: 1O9G) (Bugl et al. Reference Bugl, Fauman, Staker, Zheng, Kushner, Saper, Bardwell and Jakob2000; Mosbacher et al. Reference Mosbacher, Bechthold and Schulz2003) (Fig. 4a).

Fig. 4.

Fibrillarin resembles class I MTases. (a) Representative Class I MTase and fibrillarin structures. (b) Representative class IV MTase structures. The methylation donor, SAM, is colored in blue and the MTases are colored in yellow and green.

5.2 Pseudouridine synthases and Cbf5

In contrast to the widespread occurrence of methylation, pseudouridylation is limited to RNA molecules only. However, at least six families of pseudouridine synthases have been discovered that target one or few specific uridine(s) in each given tRNA or rRNA. Accordingly, they are named TruA, TruB, TruD, RsuA, RluA and Pus10, respectively. The Pus10 family is found in archaea and eukaryotes, but not in bacteria (McCleverty et al. Reference McCleverty, Hornsby, Spraggon and Kreusch2007). Structures of all six families of pseudouridine synthases are available and clearly indicate a conserved catalytic core among them (Hamma & Ferre-D'Amare, Reference Hamma and Ferre-D'amare2006) (Fig. 5). Crystal structures of Cbf5 have been solved either as ternary (Cbf5-Nop10-Gar1) or binary (Cbf5-Nop10) complexes (Hamma et al. Reference Hamma, Reichow, Varani and Ferre-D'amare2005; Manival et al. Reference Manival, Charron, Fourmann, Godard, Charpentier and Branlant2006; Rashid et al. Reference Rashid, Liang, Baker, Youssef, He, Phipps, Terns, Terns and Li2006) and these show that Cbf5 closely resembles TruB. In both Cbf5 and TruB, the catalytic domain comprises a nine strand β-sheet surrounded by α-helices and encompassing the active site which includes the strictly conserved aspartate. The C-terminal PseudoUridine synthase and Archaeosine transglycosylase (PUA) domain forms a mixed α/β-folding unit (Fig. 5). The PUA domain can interact with different RNA (Perez-Arellano et al. Reference Perez-Arellano, Gallego and Cervera2007), and in the case of Cbf5, it specifically interacts with the strictly conserved ACA trinucleotides of the guide RNA.

Fig. 5.

Cbf5 resembles the TruB-family of pseuoduridine synthases. Other known pseudouridylase structures are included for comparison. Key RNA-binding features are labeled where they are present. The catalytic domain is colored in light blue with a magenta catalytic aspartate and accessory domains are in green or yellow.

Stand-alone pseudouridine synthases have evolved a variety of elements to interact with the substrate RNA. These include peripheral domains for anchoring the main body of the substrate, a thumb loop, a forefinger and a motif III loop for gripping the target uridine and adjacent nucleotides (Hamma & Ferre-D'Amare, Reference Hamma and Ferre-D'amare2006). The target uridine is flipped out and tucked deep into the catalytic pocket so the catalytic residues can reach the pyrimidine base. In all enzymes, the adjacent nucleotides are remodeled in ways that depend on the RNA interaction elements in each enzyme. Dyskerin, the human Cbf5 homolog, contains extra N-terminal and C-terminal regions, but there is no structure available yet. Cbf5 lacks the forefinger and has a very short thumb loop (Fig. 5). The reduction in RNA-binding elements reflects the fact that Cbf5 functions with three other proteins and the guide RNA as discussed below.

6.1 Overview

Structural studies of box C/D RNPs have been carried out with the simpler archaeal homologs. Two crystal structures of box C/D RNPs containing all core proteins and a halfmer box C/D RNA (a single C/D module) have been obtained and one of these also contains a bound substrate RNA (Xue et al. Reference Xue, Wang, Yang, Terns, Terns, Zhang, Maxwell and Li2010; Ye et al. Reference Ye, Jia, Lin, Ju, Peng, Xu and Zhang2009). Due to limitations in crystallization, these RNP complexes are halfmer RNPs that are not active in methylation under normal in vitro experimental conditions. However, these structures have provided valuable information on protein–protein and protein–RNA interactions in an intact and functional box C/D RNP. Most significantly, when the high-resolution halfmer structural data are combined with the large body of biochemical data and an electron microscopy (EM) structure of the RNP containing full-length box C/D RNA (Bleichert et al. Reference Bleichert, Gagnon, Brown, Maxwell, Leschziner, Unger and Baserga2009), an appealing working model of box C/D function has emerged.

Each independent C/D RNP unit is organized around Nop56/58. Nop56/58 comprises an N-terminal domain, a coiled-coil domain and a CTD. Fibrillarin is bound to its N-terminal domain and this interaction is independent of RNA and L7Ae. The box C/D RNA–L7Ae complex, which forms independently of Nop56/58, is docked to the CTD of Nop56/58. Finally, the coiled-coil domain of Nop56/58 mediates self-dimerization of Nop56/58, leading to a homodimer of Nop56/58-fibrillarin-L7Ae-box C/D RNA complexes (Fig. 6a). The dyad symmetry of Nop56/58 was the basis for the initial proposal that the intact box C/D RNP has a bipartite architecture with the hairpin box C/D RNA placed along the Nop56/58 dimer (Fig. 6b) (Aittaleb et al. Reference Aittaleb, Rashid, Chen, Palmer, Daniels and Li2003; Oruganti et al. Reference Oruganti, Zhang, Li, Robinson, Terns, Terns, Yang and Li2007).

Fig. 6.

Overview of the box C/D RNP MTases containing a halfmer box C/D RNA (yellow) and a bound substrate RNA (red) in ribbon (a) and cartoon (b) representations. (PDB code: 3NMU) (a) Nop56/58 is colored in green, L7Ae is in pink and fibrillarin in light blue. SAM is colored in red. Three domains of Nop56/58 are labeled as N (N-terminal), C (C-terminal) and coiled-coli domains, respectively. The Nop56/58 helix responsible for separating the two guide strands is colored in orange. The target nucleotide is marked by a gray sphere. (b) The classic bipartite assembly model based on the halfmer RNP structure indicates that the catalytic subunits will be too far apart (90 Å) to match the potential locations of the target nucleotides (40–50 Å).

The bipartite box C/D RNP model is met with several challenges. First, the length of an intact box C/D RNA is estimated to be shorter than the length of the protein platform 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 bp with the central internal loop whose size is restricted by flanking proteins (Fig. 6b). The recent EM structure of a box C/D RNP containing an intact guide RNA revealed a surprising dual RNP model that changed the view on the traditional box C/D RNP assembly (Bleichert et al. Reference Bleichert, Gagnon, Brown, Maxwell, Leschziner, Unger and Baserga2009) and provided the basis for construction of a substrate-bound atomic model of diRNP (Xue et al. Reference Xue, Wang, Yang, Terns, Terns, Zhang, Maxwell and Li2010) (Fig. 7). In the diRNP model, the box C/D RNA is poised to bind across the two RNPs instead of along a single RNP (Fig. 7). This model alleviates the constraint on the box C/D RNA in the traditional single RNP model shown in Fig. 6b without disrupting the known protein–protein and protein–RNA interactions. The diRNP model, though, has not yet been confirmed in vivo.

Fig. 7.

The substrate-bound diRNP model of box C/D MTase (same color scheme as in Fig. 6). (a) The atomic diRNP model built from the crystal structure of the halfmer RNP is consistent with the EM density of the fully assembled diRNP (Bleichert et al. Reference Bleichert, Gagnon, Brown, Maxwell, Leschziner, Unger and Baserga2009) (EMD-1643). Fibrillarin and the N-terminal domain of Nop56/58 could not be fitted, reflecting their observed variable positioning. Single letters refer to protein subunits. ‘N’ denotes Nop56/58, ‘F’ denotes fibrillarin and ‘L’ denotes L7Ae. (b) The substrate-bound diRNP model in surface representation. (c) Cartoon representation of the substrate-bound diRNP model.

6.2 Substrate recognition

The hallmark of the box C/D RNP MTases is the stringent specificity for the target nucleotide paired with the fifth nucleotide upstream of the guide box D or D′ (n+5 rule), suggesting an importance of the first five base pairs between the guide and substrate RNAs. The only available substrate-bound RNP structure is that of a halfmer box C/D RNP bound to a 13mer substrate RNA (Xue et al. Reference Xue, Wang, Yang, Terns, Terns, Zhang, Maxwell and Li2010). In the substrate-bound structure, the guide-substrate duplex is sandwiched between a conserved Nop56/58 helix, α9A and L7Ae (Fig. 6a). The α9A helix is wedged between the guiding and non-guiding strands, creating alternating layers of protein and RNA elements. The unpaired 5′ nucleotides of the substrate RNA rest on the C-terminal end of the α9A, which further helps to direct the substrate RNA (Fig. 6a).

Surprisingly, this structure reveals that the substrate RNA is bound far from the nearest catalytic subunit fibrillarin (Fig. 6a, b) and can thus not be modified in the halfmer RNP. One solution to this problem is formation of the diRNP similar to that observed by EM (Bleichert et al. Reference Bleichert, Gagnon, Brown, Maxwell, Leschziner, Unger and Baserga2009) in which the substrate bound to one halfmer RNP may be easily placed into the active site of the opposing halfmer RNP, thereby suggesting a cross-RNP catalysis strategy (Fig. 7c). Therefore, the diRNP model provides topological solutions not only to the discrepancy in length between proteins and the guide RNA, but also to the problem of placing the substrate RNA close to the catalytic subunit.

6.3 Induced fit in box C/D RNP assembly

6.3.2 RNA kink-turn specifies the order of assembly

Another observed structural change takes place at the box C/D motif and this change is required for sequential binding of box C/D proteins to the RNA. L7Ae or its eukaryotic homolog 15·5 K binds to the box C/D motif and initiates box C/D RNP assembly (Omer et al. Reference Omer, Ziesche, Ebhardt and Dennis2002; Rashid et al. Reference Rashid, Aittaleb, Chen, Spiegel, Demeler and Li2003; Tran et al. Reference Tran, Zhang and Maxwell2003; Watkins et al. Reference Watkins, Segault, Charpentier, Nottrott, Fabrizio, Bachi, Wilm, Rosbash, Branlant and Luhrmann2000). L7Ae-box C/D RNA complex structures revealed a sharp kink in the RNA (K-turn) as a result of a tandem GA pairs and three bulged nucleotides (Fig. 8a). Biophysical studies clearly demonstrated L7Ae's ability to actively remodel the RNA (Goody et al. Reference Goody, Melcher, Norman and Lilley2004; Suryadi et al. Reference Suryadi, Tran, Maxwell and Brown2005). In contrast to RNA, L7Ae itself does not undergo conformational change and rigidly docks its conserved αβα fold onto the K-turn (Oruganti et al. Reference Oruganti, Zhang and Li2005) (Fig. 8a).

Fig. 8.

Conformational changes in box C/D RNP assembly inferred from structural studies. (a) L7Ae induces formation of kink-turn by the conserved box C/D sequences. Similar kink-turns are also observed in H/ACA RNA and U4 snRNA. (b) Multiple conformations of fibrillarin are captured in crystal structures of the RNA-free Nop56/58-fibrillarin complexes indicate a hinge motion (indicated by arrows) in Nop56/58 that can place fibrillarin.

L7Ae–K-turn interaction is not unique to box C/D RNP assembly. L7Ae is itself a ribosomal protein and has a significant sequence and structural homology to eukaryotic 15·5 K/Snu13p (box C/D and spliceosomal protein), Nhp2p (box H/ACA protein), L30 (ribosomal protein), and SBP2 that are found to interact with similar RNA motifs (Allmang et al. Reference Allmang, Carbon and Krol2002). Comparison with the available structures of L7Ae and its homologs bound with their respective substrates did not reveal obvious structural differences that explain their association with different complexes (Chao & Williamson, Reference Chao and Williamson2004; Hamma & Ferre-D'Amare, Reference Hamma and Ferre-D'amare2004; Moore et al. Reference Moore, Zhang, Fenley and Li2004; Vidovic et al. Reference Vidovic, Nottrott, Hartmuth, Luhrmann and Ficner2000) (Fig. 8a). It is possible that differences in the dynamics of binding contribute to their physiological specificity. In higher-order complexes, as illustrated in the box C/D and box H/ACA RNP assemblies, specificity is further defined by subsequently interacting proteins.

7.1 Overview

Studies on H/ACA RNP structures have also been carried out with archaeal homologs reconstituted from recombinant and synthetic components. These studies not only provide molecular details for understanding the biochemical mechanisms of H/ACA RNPs, but also shed light on the assembly architecture of hTR holoenzyme.

In vitro biochemical studies of box H/ACA RNPs reveal that three of the four proteins are essential to pseudouridylation, while the fourth protein, Gar1, significantly improves the enzyme activity (Baker et al. Reference Baker, Youssef, Chastkofsky, Dy, Terns and Terns2005; Charpentier et al. Reference Charpentier, Muller and Branlant2005). Both Cbf5 and L7Ae interact with box H/ACA guide RNAs directly, while Nop10 and Gar1 interact with the guide RNAs indirectly through their interactions with Cbf5 (Baker et al. Reference Baker, Youssef, Chastkofsky, Dy, Terns and Terns2005; Charpentier et al. Reference Charpentier, Muller and Branlant2005). Structures of the box H/ACA RNP, especially those containing substrate RNA, provide the structural basis for these biochemical observations.

The structure of a substrate-free archaeal box H/ACA RNP containing all four proteins, Cbf5, Nop10, Gar1 and L7Ae and a single-hairpin H/ACA RNA revealed the first complete view of box H/ACA RNP architecture (Fig. 9) (Li & Ye, Reference Li and Ye2006). Cbf5 is the master organizer that interacts extensively with the guide RNA, Nop10 and Gar1. Nop10 is a small protein, containing an N-terminal zinc binding domain of four packed β-sheets, a C-terminal single helix domain and a linker loop. Nop10 is anchored against the catalytic domain of Cbf5. This interaction is required for its stabilization because Nop10 is significantly unfolded when in isolation (Hamma et al. Reference Hamma, Reichow, Varani and Ferre-D'amare2005). Gar1 adopts a six β-strand barrel fold that is similar to that found in some RNA-binding proteins such as the translation elongation factor EF-Tu (Rashid et al. Reference Rashid, Liang, Baker, Youssef, He, Phipps, Terns, Terns and Li2006). One study has found that Gar1 interacts with snoRNA directly in vitro (Bagni & Lapeyre, Reference Bagni and Lapeyre1998) and a cross-link study placed mammalian Gar1 close to the substrate RNA (Wang & Meier, Reference Wang and Meier2004). In the crystal structure of the box H/ACA RNP, it, however, binds to one side of Cbf5 that is away from the RNA. The guide RNA lies linearly along the protein surface comprised of Cbf5, Nop10 and L7Ae without contacting Gar1. The two ends of the box H/ACA RNA are secured by two specific RNA–protein interactions. The conserved ACA sequence of the guide RNA at the 3′-end are recognized specifically by the PUA domain of Cbf5 (Fig. 9) and the apical K-loop is recognized in the same manner as the box C/D motif by L7Ae (Fig. 8a). L7Ae also interacts with Cbf5-bound Nop10, which brings the upper stem of the guide RNA in close contact with Cbf5 and Nop10. Comparison of crystal structures of either binary or ternary protein complexes reveals no gross conformational changes in protein upon association with the guide RNA (Rashid et al. Reference Rashid, Liang, Baker, Youssef, He, Phipps, Terns, Terns and Li2006), suggesting that proteins are rigidly docked to the guide RNA.

Fig. 9.

Overall view of the substrate-bound archaeal box H/ACA RNP (PDB code: 2HVY). Each subunit is labeled. The specific interaction between the trinucleotide ACA and the PUA domain of Cbf5 is highlighted.

7.2 Substrate recognition

Significant details are revealed by substrate-bound RNP structures containing different H/ACA proteins (Duan et al. Reference Duan, Li, Lu, Wang and Ye2009; Liang et al. Reference Liang, Xue, Terns, Terns and Li2007a, Reference Liang, Zhou, Kahen, Terns, Terns and Li2009a). Comparing these with the stand-alone pseudouridine synthase–RNA complexes (Alian et al. Reference Alian, Degiovanni, Griner, Finer-Moore and Stroud2009; Hoang et al. Reference Hoang, Chen, Vizthum, Kandel, Hamilton, Mueller and Ferre-D'amare2006; Hoang & Ferre-D'Amare, Reference Hoang and Ferre-D'amare2001; Hur & Stroud, Reference Hur and Stroud2007; Pan et al. Reference Pan, Agarwalla, Moustakas, Finer-Moore and Stroud2003) provides even greater insight. In all these studies, the target uridine is substituted by 5-fluorouridine, which the enzyme converts to 5-fluoro, 6-hydroxypseudouridine (5FhΨ). It is believed that extraction of the fluorine atom (or proton in uridine) and the C6 hydroxyl from 5FhΨ would lead to the final isomerized product. The gross features of enzyme–substrate interactions observed in this reaction intermediate complex are expected to remain throughout the reaction. However, detailed interactions between the substrate and the active site residues are expected to vary at different stages of the reaction.

Box H/ACA RNPs recruit substrates through base pair interactions between the guide and the substrate RNA. By pairing with both strands of the internal loop, the substrate RNA forms a three-way junction (Ω-like motif) with the guide RNA (Fig. 10a). This Ω-like motif is unconstrained in the absence of proteins (Jin et al. Reference Jin, Loria and Moore2007; Wu & Feigon, Reference Wu and Feigon2007) but significantly remodeled when bound to proteins such that the target uridine and two adjacent nucleotides are sharply kinked (Duan et al. Reference Duan, Li, Lu, Wang and Ye2009; Liang et al. Reference Liang, Xue, Terns, Terns and Li2007a; Liang et al. Reference Liang, Zhou, Kahen, Terns, Terns and Li2009a) (Fig. 10a). The 3′ guide–substrate duplex mimics the TΨC stem of tRNA recognized by TruB. Like TruB, the thumb loop (β7_10 loop) interacts with the major groove of the stem and the motif III loop catches the approaching substrate kink (Fig. 10a). The substituted target nucleotide is converted to 5FhΨ and secured deep inside the active site pocket by a network of interactions with the catalytic residues (Fig. 10a) (Duan et al. Reference Duan, Li, Lu, Wang and Ye2009; Liang et al. Reference Liang, Zhou, Kahen, Terns, Terns and Li2009a). In both TruB and box H/ACA RNP, the thumb loop is open in the absence of substrate RNA but is closed in its presence (Duan et al. Reference Duan, Li, Lu, Wang and Ye2009; Hoang et al. Reference Hoang, Hamilton, Mueller and Ferre-D'amare2005; Liang et al. Reference Liang, Zhou, Kahen, Terns, Terns and Li2009a). The short thumb loop of Cbf5 interacts only with one nucleobase, that of the +1 nucleotide, and two phosphate groups, those of the +1 and −4 nucleotides (the target nucleotide is assumed to be position 0) (Fig. 10a).

Fig. 10.

Substrate binding to box H/ACA RNP. (a) Overview of the substrate-bound box H/ACA RNP (PDB code: 3HJW or 3HAX). The asterisk indicates that Gar1 is modeled for the substrate-bound complex according its position in substrate-free RNP complex (PDB code: 2HVY) or that of the low-resolution structure of the fully assembly complex (PDB code: 3HAY). Interactions between the target nucleotide (5FhΨ) and the two flanking nucleotides are highlighted in the right upper panel. Protein residues are labeled according to Pyrococcus furiosus Cbf5 numbering. The right lower panel indicates the open and closed conformations of the β7_10 loop (thumb loop) of Cbf5 that accompany docking of the substrate RNA. (b) Comparison of guide-target RNA duplexes in protein-free (PDB code: 2PCW and 2P89), all protein- (+L7Ae) (PDB code: 3HJW), or those without L7Ae (−L7Ae)-bound (PDB code: 2RFK) states.

Owing to its dependence on the guide RNA, the RNA substrate of a box H/ACA RNP is therefore also influenced by proteins beyond the catalytic subunit. This is best illustrated by comparing the substrate-bound RNP structures and substrate fluorescence intensities in the presence and absence of L7Ae or Gar1 (Duan et al. Reference Duan, Li, Lu, Wang and Ye2009; Liang et al. Reference Liang, Zhou, Kahen, Terns, Terns and Li2009a). L7Ae interacts with the upper stem of the guide RNA adjacent to Nop10. In the absence of L7Ae, the upper stem is released from its anchored position, which leads to the departure of the substrate RNA from the active site (Liang et al. Reference Liang, Xue, Terns, Terns and Li2007a) (Fig. 10b).

The structural effect of Gar1 on substrate binding is not only more subtle, but also complex. The absence of Gar1 only slightly shifts the upper stem of the guide RNA (Liang et al. Reference Liang, Zhou, Kahen, Terns, Terns and Li2009a). Fluorescence studies on substrate docking showed that addition of Gar1 to the otherwise fully assembled RNP caused an initial undocking followed by redocking of the substrate RNA (Liang et al. Reference Liang, Kahen, Calvin, Zhou, Blanco and Li2008). These observations are consistent with the non-essential but accelerating role of Gar1 in pseudouridylation reactions (Baker et al. Reference Baker, Youssef, Chastkofsky, Dy, Terns and Terns2005; Charpentier et al. Reference Charpentier, Muller and Branlant2005; Muller et al. Reference Muller, Fourmann, Loegler, Charpentier and Branlant2007). The fact that Gar1 does not directly contact either the guide or the substrate RNA indicates that Gar1 functions through its interaction with Cbf5, especially the Cbf5 thumb loop.

The flexibility of the guide RNA in directing substrate binding and modification has also been investigated. Comparing secondary structures of the available H/ACA guide RNAs indicates that the distance between the target uridine and the ACA trinucleotide may vary as a result of different numbers of paired bases in the 3′ substrate-guide helix (SH1) or in the guide RNA lower stem (P1) (Figs 1 and 10a). Comparing structures having two different distances (14 and 15 bp) in the P1 and SH1 revealed virtually no difference around the target uridine (Duan et al. Reference Duan, Li, Lu, Wang and Ye2009; Liang et al. Reference Liang, Zhou, Kahen, Terns, Terns and Li2009a). This indicates that the guide RNA is able to adjust its two junctions (J1 and J2, Fig. 10a) in order to correctly place the substrate RNA. Furthermore, mutational studies with altered guide RNA in its pseudouridine pocket, thus different SH1, and its lower stem P1 suggests an even wider range of guide flexibility in these regions (Liang et al. Reference Liang, Zhou, Kahen, Terns, Terns and Li2009a).

7.3 Pre-reactive, intermediate and post-reactive structures

In searching for the active site geometries at other stages of the isomerization reaction, crystal structures of the box H/ACA RNPs bound with substrate RNA containing various uridine analogs were obtained (Zhou et al. Reference Zhou, Liang and Li2010a, Reference Zhou, Chao, Liang, Chen, Yang and Lib). These include two pre-reactive complexes containing 5-bromouridine (5BrU) and 3-methyluridine (3MU) and two product complexes containing 2-deoxyuridine (2dU) and 4-thiouridine (4SU) in place of the target uridine. The two product nucleotides, 2dU and 4SU, are not found in the active site, while the flanking nucleotides are, suggesting their low affinities for the enzyme. The two pre-reactive complex structures show that the substituted uridine residues are well ordered in the active site and that they bind less deep within the active site than 5FhΨ (Fig. 11, 5BrU, 3MU and 5FhΨ). Interestingly, in these complexes of different reaction status, the catalytic aspartate is oriented in a similar fashion with respect to the anomeric carbon C1′ but not to ring carbon C6. This result supports the possible role of C6 in catalysis as previously proposed (Gu et al. Reference Gu, Liu and Santi1999).

Fig. 11.

Toward understanding of the catalytic steps of pseudouridylation. Substitution of C5 hydrogen by bromide (5BrU) or N3 hydrogen by methyl group (3MU) inhibited uridine isomerization while substitution of C5 hydrogen by fluoride did not inhibit the ring cleavage step, leading to 5,6-hydroxy-pseudouridine (5FhΨ) intermediate. Superimposed crystal structures of the Cbf5 active site with bound 5FhΨ-, 5BrU- and 3MU-substituted substrates (PDB codes: 3HJW, 3LWO and 3LWQ, respectively) indicate that the catalytic aspartate is positioned similarly to the anomeric carbon C1′ but dissimilarly to ring carbon C6, suggesting an important role of C6 in catalysis. Protein residues are labeled according to Pyrococcus furiosus Cbf5 numbering and colored in grey, blue and red. The target nucleotide is colored in red.

The structures of different reaction stages also reveal remodeling of the thumb loop of Cbf5. The thumb loop is disordered in the pre-reactive and product complexes but stabilized in the 5FhΨ complex, suggesting its distinct role in binding the reaction intermediate. The thumb loop of TruB is already known to take different positions in structures containing either catalytically deficient TruB complex or containing 5FhΨ (Hoang et al. Reference Hoang, Hamilton, Mueller and Ferre-D'amare2005).

7.4 Structures of box H/ACA sno/scaRNP assembly factors

Shq1 and Naf1 are two conserved trans-acting factors involved in assembly of box H/ACA sca/snoRNPs. They bind early to Cbf5 during co-transcriptional H/ACA RNP assembly and are released from the mature RNPs. Structures of the N-terminal domain of Shq1 (PDB codes: 3EUD and 2K8Q) and the GAR1 domain of Naf1 (PDB code: 2V3M) have been solved (Godin et al. Reference Godin, Walbott, Leulliot, van Tilbeurgh and Varani2009; Leulliot et al. Reference Leulliot, Godin, Hoareau-Aveilla, Quevillon-Cheruel, Varani, Henry and Van Tilbeurgh2007; Singh et al. Reference Singh, Gonzales, Cascio, Heckmann, Chanfreau and Feigon2009). Superimposing Shq1 and Naf1 to Gar1 reveals a strikingly similar four β-strand sheet among these three proteins (Fig. 12). Gar1 uses this motif to interact with Cbf5. This structural comparison supports the proposed model of competition between Gar1 and Shq1/Naf1 for binding to Cbf5 in the late stages of box H/ACA RNP assembly (Darzacq et al. Reference Darzacq, Kittur, Roy, Shav-Tal, Singer and Meier2006; Godin et al. Reference Godin, Walbott, Leulliot, van Tilbeurgh and Varani2009; Grozdanov et al. Reference Grozdanov, Roy, Kittur and Meier2009).

Fig. 12.

Biogenesis factors of box H/ACA RNPs share structural homologies with Gar1.