ACA Small Ribonucleoproteins

Molecular Cell

Review Box H/ACA Small Ribonucleoproteins Tama´s Kiss,1,2,* Ele´onore Fayet-Lebaron,1 and Bea´ta E. Ja´dy1 1Laboratoire

de Biologie Mole´culaire Eucaryote du CNRS, UMR5099, IFR109 CNRS, Universite´ Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse Cedex 9, France 2Biological Research Centre, Hungarian Academy of Sciences, 62 Temesva ´ ri krt, 6726 Szeged, Hungary *Correspondence: [email protected] DOI 10.1016/j.molcel.2010.01.032

Box H/ACA RNAs represent an abundant, evolutionarily conserved class of small noncoding RNAs. All H/ACA RNAs associate with a common set of proteins, and they function as ribonucleoprotein (RNP) enzymes mainly in the site-specific pseudouridylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Some H/ACA RNPs function in the nucleolytic processing of precursor rRNA (pre-rRNA) and synthesis of telomeric DNA. Thus, H/ACA RNPs are essential for three fundamental cellular processes: protein synthesis, mRNA splicing, and maintenance of genome integrity. Recently, great progress has been made toward understanding of the biogenesis, intracellular trafficking, structure, and function of H/ACA RNPs. Introduction Box H/ACA RNAs are one of the largest and evolutionarily most conserved families of small noncoding RNAs that are present in all eukaryotes and archaea (for recent reviews, see Kiss et al., 2006; Matera et al., 2007; Meier, 2005; Terns and Terns, 2006). The H/ACA RNAs function mainly as guide RNAs in the sitespecific synthesis of pseudouridines in rRNAs, spliceosomal small nuclear RNAs (snRNAs), and probably in other classes of cellular RNAs. Pseudouridine, a C5-glycoside isomer of uridine, is the most common posttranscriptionally synthesized modified ribonucleotide in cellular RNAs (Charette and Gray, 2000) (Figure 1A). Pseudouridines in rRNAs and snRNAs are essential for the correct function of the ribosome and spliceosome (Yu et al., 2005). In higher eukaryotes, H/ACA RNAs directing rRNA pseudouridylation (snoRNAs) accumulate in the nucleolus, whereas H/ACA RNAs mediating pseudouridylation of spliceosomal snRNAs (small Cajal body-specific RNAs, scaRNAs) reside in the nucleoplasmic Cajal bodies (CBs) (Darzacq et al., 2002). Archaeal H/ACA RNAs are usually composed of a single 60–75 nt long hairpin, and most eukaryotic H/ACA RNAs contain two hairpins which are followed by the single-stranded H (AnAnnA) and ACA box motifs (Figure 1B) (Balakin et al., 1996; Dennis and Omer, 2005; Ganot et al., 1997b). The bipartite pseudouridylation-guide sequences are located in the distal (upper) part of an internal loop, called the pseudouridylation pocket, of the 50 - or 30 -terminal hairpin of the guide RNA (Ganot et al., 1997a) (Figure 1C). Frequently, both hairpins of the H/ACA guide RNA carry functional pseudouridylation pockets that can direct pseudouridylation of distantly located uridines in the precursor rRNA (pre-rRNA). In the guide RNA-target RNA interaction, the unpaired substrate uridine and its 50 -neighboring nucleotide are positioned at the base of the upper stem closing the pseudouridylation pocket. The distance between the selected uridine and the H or ACA box of the guide RNA is about 14 nt. Vertebrate H/ACA scaRNAs also carry a CB localization signal (CAB box, with a consensus of ugAG) in the terminal loop of their 50 - and/ or 30 -hairpins (Richard et al., 2003). Archaeal H/ACA RNAs contain an additional protein-binding motif, the kink- (k)-turn,

that is also present in box C/D RNAs, another class of noncoding RNAs directing 20 -O-methylation of rRNAs and snRNAs (Rozhdestvensky et al., 2003). To form functionally active pseudouridylation-guide ribonucleoproteins (RNPs), H/ACA RNAs associate with four evolutionarily conserved RNP proteins: Cbf5 (dyskerin in human, Nap57 in rodents), Nhp2 (L7Ae in archaea), Nop10, and Gar1. For the H/ACA RNP holoenzyme, the Cbf5/dyskerin pseudouridine synthase provides the catalytic activity (Lafontaine et al., 1998; Zebarjadian et al., 1999). Although RNA pseudouridylation is the major, and most likely the ancestral, function of H/ACA RNPs, some members of the family acquired new functions during evolution. Two yeast H/ACA snoRNAs, snR30 and snR10, function in the nucleolytic processing of pre-rRNA, and the vertebrate telomerase H/ACA RNA directs the synthesis of telomeric DNA (Bally et al., 1988; Collins, 2006; Morrissey and Tollervey, 1993; Tollervey, 1987). During the past years, dozens of H/ACA RNAs of unknown function have been described from various organisms, suggesting that the functional repertoire of H/ACA RNAs will be extended in the future. In this review, we will focus on recent advances concerning the structural organization, biogenesis, and functional diversity of H/ACA RNPs. Evolution of the H/ACA Pseudouridylation-Guide Machinery In Eubacteria, synthesis of pseudouridines is mediated exclusively by protein enzymes that lack RNA cofactors (Hamma and Ferre-D’Amare, 2006). The RNA-guided RNA pseudouridylation mechanism was apparently established in the common ancestor of archaea and eukarya about 2–3 billion years ago (Kuhn et al., 2002; Omer et al., 2000). It is likely that primordial pseudouridylation-guide RNAs evolved from cis-acting rRNA or tRNA guide sequences. Consistent with this, the archaeal L7Ae ribosomal protein, besides binding to the 23S rRNA, is an integral component of box H/ACA and C/D RNPs, suggesting that both classes of modification guide RNPs evolved from an rRNA-derived ancestor (Kuhn et al., 2002; Rozhdestvensky et al., 2003). On the other hand, the H/ACA RNA-associated pseudouridine synthase Cbf5/dyskerin shows strong structural

Molecular Cell 37, March 12, 2010 ª2010 Elsevier Inc. 597

Molecular Cell

Review pseudouridines present in Escherichia coli rRNAs, vertebrate rRNAs and snRNAs together contain about 130 H/ACA RNPsynthesized pseudouridines, which are located in diverse sequence and structural environments.

Figure 1. Structure and Function of H/ACA RNAs (A) Isomerization of uridine to pseudouridine. After breakage of the N1-C1 bond of uridine, the free uracil is rotated 180 around the N3-C6 axis and reattached to the ribose through a C1-C5 bond. The resulting pseudouridine possesses an additional potential hydrogen bond donor (N1, indicated in yellow). (B) Schematic structure of archaeal and eukaryotic H/ACA RNAs. The conserved H, ACA, CAB, and k-turn motifs are shown. The target recognition sequences in the pseudouridylation loop (J pocket) are in brown. (C) Selection of target uridines by H/ACA pseudouridylation guides RNAs.

similarity to the bacterial pseudouridine synthase TruB that is responsible for the synthesis of the conserved J55 in the T loop of bacterial tRNAs (Hoang and Ferre-D’Amare, 2001). Bioinformatic searches have shown that eukaryotic H/ACA RNA genes are highly mobile genetic elements that are frequently duplicated by retrotransposition (Luo and Li, 2007; Weber, 2006). Duplication of H/ACA RNA genes followed by spontaneous mutations of the target recognition sequences can give rise to novel pseudouridylation-guide RNAs. Thus, the RNA-guided RNA pseudouridylation system seems to have an inherent ability for rapid evolution. Indeed, in contrast to the 11

598 Molecular Cell 37, March 12, 2010 ª2010 Elsevier Inc.

Structural and Functional Organization of Archaeal H/ACA RNPs Although box H/ACA pseudouridylation-guide RNAs and RNP proteins have been extensively characterized in many evolutionarily distant species, including vertebrates, yeasts, plants, and archaea, the structural organization of H/ACA RNPs has remained largely unknown until recently. Understanding the structure of H/ACA RNPs has been facilitated by the development of in vitro systems for reconstitution of enzymatically active archaeal pseudouridylation-guide RNPs by incubation of in vitrosynthesized archaeal single-hairpin H/ACA RNAs with purified recombinant Cbf5, L7Ae, Nop10, and Gar1 proteins (Baker et al., 2005; Charpentier et al., 2005). These experiments demonstrated that both Cbf5 and L7Ae bind to H/ACA RNAs directly and independently from other H/ACA proteins (Figure 2A). L7Ae binds to the k-turn of archaeal H/ACA RNAs, and it fails to interact with other H/ACA proteins in the absence of the guide RNA. Cbf5 binds to H/ACA RNAs in an ACA box-dependent manner, but in contrast to L7Ae, it also interacts with Gar1 and Nop10 in an RNA-independent manner to form a stable Cbf5Gar1-Nop10 heterotrimeric H/ACA subcomplex. Although a subcomplex containing the guide RNA, Cbf5, and Nop10 displays basal level of pseudouridylation activity, all four H/ACA proteins are necessary for efficient pseudouridylation of the substrate RNA by Cbf5. The Cbf5-Nop10 dimeric and the Cbf5-Nop10-Gar1 trimeric protein subcomplexes of archaeal H/ACA RNPs have been crystallized and their structures determined (Hamma et al., 2005; Manival et al., 2006; Rashid et al., 2006). As predicted by their sequence similarities (Koonin, 1996), Cbf5 showed a strong structural similarity to the E. coli pseudouridine synthase TruB. Cbf5 features both the N-terminal catalytic domain and the C-terminal pseudouridine synthase and archaeosine transglycosylase (PUA) domain of TruB (Hoang and Ferre-D’Amare, 2001; Pan et al., 2003). The active sites of Cbf5 and TruB are structurally highly similar. For example, the catalytic aspartate (Asp85) of Cbf5 that is conserved among all pseudouridine synthases perfectly superimposes in space with the catalytic Asp48 of E. coli TruB. This suggests that Cbf5 and TruB use the same ‘‘base-flipping’’ mechanism to convert uridine to pseudouridine (Hamma and Ferre-D’Amare, 2006). Nop10 and Gar1 bind to two distant orthogonal faces of the D1 and D2 subdomains of the catalytic domain of Cbf5, confirming that they interact with Cbf5 independently of each other (Hamma et al., 2005; Manival et al., 2006; Rashid et al., 2006) (Figure 2B). Nop10 adopts an extended structure composed of an N-terminal b-ribbon zinc-binding motif, a flexible but highly conserved linker region, and a short C-terminal a helix. The linker region of Nop10 tightly interacts with a conserved stretch of Cbf5 that is in close vicinity to the catalytic site and that plays an important role in the stability of other pseudouridine synthases, indicating that Nop10 may contribute to the structural organization and stabilization of the catalytic center of Cbf5. Although Gar1 forms

Molecular Cell

Review

Figure 2. Structure and Function of Archaeal H/ACA RNPs (A) Organization of archaeal H/ACA pseudouridylation-guide RNPs. The H/ACA RNP proteins and the RNA structural elements are indicated by color code. (B) Crystal structure of archaeal H/ACA RNP. The front (left) and side (right) views are shown. The star indicates the catalytic center of Cbf5. Reproduced with permission from Li and Ye (2006). (C) Positioning of the recruited substrate RNA in an H/ACA RNP in the absence of Gar1. The substrate RNA bound to the pseudouridylation pocket forms a U-shaped structure that is aligned vertically to the active surface of Cbf5. Reproduced with permission from Duan et al. (2009). (D) Substrate-induced conformational switch of the thump loop of Cbf5. The Gar1-associated open (gray) and the substrate RNA-bound closed (green) conformations of the thumb loop are shown. The residues involved in protein-protein interactions are highlighted. Reproduced with permission from Duan et al. (2009).

a six-stranded b barrel reminiscent of the RNA-binding motifs of bacterial translation initiation (IF2) and elongation (IF-G) factors, the crystal structure of the archaeal Cbf5-Gar1-Nop10 core complex strongly argues against a direct role for Gar1 in H/ ACA guide RNA or substrate RNA binding. Instead, the putative RNA-binding elements of Gar1 are engaged in formation of direct contacts with an RNA-binding loop of Cbf5, called the thumb loop (see below), indicating that Gar1 modulates the RNA-binding capacity of Cbf5 (Li and Ye, 2006; Rashid et al., 2006). Shortly after the structure of the Cbf5-Nop10-Gar1 complex was solved, the crystal structure of the entire archaeal H/ACA RNP was solved (Li and Ye, 2006). The archaeal single-hairpin H/ACA RNA is composed of the proximal stem (P1) followed by the ACA box, the pseudouridylation pocket, and the distal stem (P2), with the k-turn adopting a slightly bended structure (Figure 2B). The ACA box and the P1 stem are bound to the PUA domain of Cbf5, while the distal P2 stem is tightly fastened to a composite surface formed by Nop10, L7Ae, and the D1 subdomain of Cbf5 (Hamma and Ferre-D’Amare, 2004; Li and Ye, 2006; Liang et al., 2007). The bipartite interaction of the Cbf5-Nop10-L7Ae complex with the guide RNA positions the pseudouridylation pocket near the active cleft of Cbf5. In contrast to the P1 and P2 stems, the pseudouridylation loop nucleotides form very few intermolecular contacts with H/ACA

proteins; instead they show a great flexibility in the substratefree RNP (Li and Ye, 2006). The high level of structural conservation of H/ACA proteins indicates that eukaryotic and archaeal H/ACA RNPs share a very similar architecture (Manival et al., 2006), except that eukaryotic H/ACA RNAs contain two hairpins which bind two sets of H/ACA proteins (Watkins et al., 1998). Similarly to archaeal Cbf5, yeast and mammalian Cbf5 (the latter is called dyskerin in human and Nap57 in rodents) interacts with Nop10 and Gar1 and at least yeast Cbf5 directly binds to the H/ACA RNA (Henras et al., 2004; Normand et al., 2006; Wang and Meier, 2004). Eukaryotic H/ACA RNAs lack k-turn motifs, and Nhp2, the eukaryotic homolog of L7Ae, is recruited to H/ACA RNPs through protein-protein interactions. Mammalian Nhp2 binds to the preformed dyskerin-Nop10 complex to form a stable trimeric H/ACA subcomplex (Wang and Meier, 2004). The pseudouridylation loop nucleotides of H/ACA RNAs can base pair with substrate RNAs both in the absence and presence of H/ACA proteins (Jin et al., 2007; Liang et al., 2007; Wu and Feigon, 2007). In the guide RNA-substrate RNA complex, the substrate RNA interacts on one face of the guide RNA, resulting in an U-shaped interaction. Recently, the crystal structures of substrate-bound archaeal H/ACA RNPs with or without Gar1 have been solved (Duan et al., 2009; Liang et al., 2009). The overall shape of the guide RNA in the substrate-bound RNP is similar

Molecular Cell 37, March 12, 2010 ª2010 Elsevier Inc. 599

Molecular Cell

Review

Figure 3. Biogenesis of Eukaryotic H/ACA RNPs Is Promoted by Multiple Trans-Acting Factors For simplicity reasons, only one H/ACA hairpin is shown. The known interactions of H/ACA core proteins and H/ACA assembly factors are shown. The precise molecular role of most assembly factors remains to be elucidated. For details, see the text.

to that observed for the free RNP. However, docking of the substrate RNA induces a conformational reorganization of the pseudouridylation pocket and an adaptive movement of the proximal P2 stem of the guide RNA together with the PUA domain of Cbf5. Correct positioning of the distal (P1) and proximal (P2) stems of the guide RNA not only places the bound substrate RNA near the catalytic cleft of Cbf5 but also facilitates its folding into a catalytic-competent conformation (Figure 2C). The substrate-bound H/ACA RNA-Cbf5 complex shares striking structural similarities with the previously characterized TruB-tRNA complex (Duan et al., 2009; Hoang and FerreD’Amare, 2001). The H/ACA P1 stem followed by the ACA motif is structurally related to the tRNA acceptor stem followed by the CCA motif,and these motifs bind to the PUA domain of Cbf5 and TruB, respectively. The short helix formed by the 30 guide sequence of the pseudouridylation pocket and the bound substrate RNA interacts with the catalytic domain of Cbf5 (Figure 2C). Likewise, the T stem of the tRNA binds to the catalytic domain of TruB. Correct docking of these rigid helical stems is instrumental for placing the unpaired substrate uridine that immediately follows these helices to the catalytic center of Cbf5 and TruB. In the substrate-bound H/ACA RNP and TruBtRNA complex, the analogous RNA and protein elements superpose in space. The striking structural similarity of the archaeal H/ACA RNP and the eubacterial TruB-tRNA complex has been further underlined by the finding that archaeal Cbf5 can catalyze the in vitro synthesis of J55 in tRNA in the absence of guide RNA (Roovers et al., 2006). Cbf5 and TruB share a common Arg-rich RNA-binding loop, the thumb loop, that is often present in other pseudouridine synthases, and this thumb loop contributes to specific substrate recognition (Hamma and Ferre-D’Amare, 2006). The thumb loop of Cbf5 seems to play a central role in the recruitment and release of H/ACA substrate RNAs (Figure 3D). In the substrate-

600 Molecular Cell 37, March 12, 2010 ª2010 Elsevier Inc.

free H/ACA RNP, the thumb loop adopts an ‘‘open’’ conformation, and instead of binding to the guide RNA, it associates with Gar1 (Duan et al., 2009; Li and Ye, 2006; Liang et al., 2007). In the substrate-bound H/ACA RNP, however, the thumb loop interacts with the U-shaped substrate RNA and locks the substrate uridine (Duan et al., 2009; Liang et al., 2009). Consistent with the sequence diversity of the cognate substrate RNAs of H/ACA RNPs, the thumb loop forms nonspecific hydrogen bonding and electrostatic contacts with the sugar-phosphate backbone of the recruited substrate RNA. Thus, the observed conformational switch of the thumb loop of Cbf5 between Gar1 and the substrate RNA seems to be the central step in controlling the substrate turnover in the H/ACA RNP-mediated pseudouridylation reaction. In conclusion, solving of the crystal structure of fully assembled archaeal H/ACA RNPs bound to their substrate RNAs provided valuable insights into the molecular roles of H/ACA core proteins and guide RNAs played in the H/ACA RNP-mediated RNA pseudouridylation reaction. Biogenesis of Eukaryotic H/ACA RNPs Considering that functional archaeal H/ACA RNPs can be reconstituted in vitro, biogenesis of eukaryotic H/ACA RNPs is a surprisingly complex process that requires multiple accessory factors (Kiss et al., 2006; Terns and Terns, 2006; Matera et al., 2007). In archaea and lower eukaryotes, the H/ACA RNA genes are most frequently organized in independent mono- or polycistronic transcription units, whereas in higher eukaryotes there is a strong tendency toward intronic integration of H/ACA RNA coding units. As a consequence, all vertebrate H/ACA RNAs, with the exception of telomerase RNA, are encoded in premRNA introns. Assembly of yeast H/ACA precursor RNPs (preRNPs) occurs during transcription of the nascent H/ACA RNAs (Ballarino et al., 2005; Yang et al., 2005). Likewise, recognition of mammalian intronic H/ACA RNA sequences and recruitment

Molecular Cell

Review of H/ACA RNP proteins occurs on the newly synthesized host pre-mRNA in parallel with splice site selection (Darzacq et al., 2006; Richard et al., 2006). The most extensively characterized H/ACA assembly factors, Naf1 and Shq1, function only in the biogenesis of H/ACA RNPs. Naf1 and Shq1 are nucleocytoplasmic shuttle proteins, and they accumulate in the nucleoplasm but are excluded from the nucleoli and CBs where functional H/ACA RNPs reside (Darzacq et al., 2006; Dez et al., 2002; Grozdanov et al., 2009b; Kittur et al., 2006; Yang et al., 2002). Both Naf1 and Shq1 bind to Cbf5/dyskerin probably to provide metabolic stability for this inherently instable protein and to facilitate its correct subcellular localization (Darzacq et al., 2006; Dez et al., 2002; Fatica et al., 2002; Godin et al., 2009; Yang et al., 2002) (Figure 3). While Naf1 interacts mainly with the dyskerin-Nop10-Nhp2 H/ACA core complex, Shq1 binds only to the free Cbf5/dyskerin, suggesting that Shq1 functions in an early step in H/ACA RNP biogenesis and before Naf1. Shq1 may directly bind to the newly synthesized Cbf5/dyskerin and may accompany it until the association with Nop10 and Nhp2. Besides interacting with the dyskerin-Nop10-Nhp2 H/ACA core complex, Naf1 also binds to the phosphorylated carboxyterminal domain of RNA polymerase (Pol II) and interacts with Pol II-specific transcription factors (Fatica et al., 2002; Yang et al., 2005). Therefore, Naf1 may target the dyskerin-Nop10Nhp2 complex to the newly synthesized H/ACA RNAs through interacting with Pol II. Supporting this idea, Naf1, Cbf5/dyskerin, Nop10, and Nhp2 specifically accumulate at actively transcribed yeast and mammalian H/ACA RNA genes (Ballarino et al., 2005; Darzacq et al., 2006; Yang et al., 2005). The nascent precursor H/ACA RNAs interact with the preformed Naf1-dyskerinNop10-Nhp2 complex to form inactive pre-H/ACA RNPs which still lack Gar1 (Ballarino et al., 2005; Darzacq et al., 2006; Yang et al., 2005). A domain of yeast Naf1 forms a six-stranded b barrel fold similar to the Cbf5-binding domain of archaeal Gar1 (Leulliot et al., 2007). Using these common domains, Naf1 and Gar1 bind to the same site of Cbf5/dyskerin in a mutually exclusive manner (Darzacq et al., 2006; Wang and Meier, 2004). The last step of H/ACA RNP assembly is the replacement of Naf1 with Gar1, which controls the transition from inactive pre-RNP to functional H/ACA RNP (Darzacq et al., 2006). The survival of motor neurons (SMN) complex that promotes assembly of various classes of RNPs has been shown to specifically interact with Gar1, suggesting that SMN also functions in H/ACA RNP assembly (Matera et al., 2007). Given that the nuclear fraction of SMN concentrates in CBs and that nascent box C/D and H/ACA RNPs are targeted to the CBs by the snRNA export adaptor PHAX, it seems possible that the SMN-mediated incorporation of Gar1 occurs in the CB (Boulon et al., 2004). The heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that, in collaboration with a variety of cochaperones and cofactors, promotes the proper folding of important signaling factors (Pearl and Prodromou, 2006). Recently, Hsp90 has been demonstrated to function in the assembly of eukaryotic RNPs containing the L7Ae-related Nhp2 (box H/ACA RNPs), 15.5K (box C/D RNPs), and Sbp2 (selenoprotein mRNPs) RNP proteins (Boulon et al., 2008; Venteicher et al., 2008; Zhao et al., 2008). Interestingly, the N-terminal part of the H/ACA

assembly factor Shq1 is structurally homologous to the CHORD-containing proteins and Sgt1 (CS) domain, which is present in a number of cochaperones for Hsp90 (Godin et al., 2009; Singh et al., 2009). The CS domain of Shq1 is essential for the biogenesis of H/ACA RNPs, but in contrast to the CS domains of authentic Hsp90 cochaperones, it fails to interact with Hsp90 (Godin et al., 2009; Singh et al., 2009). In the biogenesis of the L7Ae RNP family, Hsp90 functions together with the R2TP complex that is composed of four proteins: Rvb1 (also called Pontin and Tip49), Rvb2 (also called Reptin and Tip48), Tah1 (Spagh in human), and Pih1 (also called Nop17) (Boulon et al., 2008; Zhao et al., 2005, 2008). Rvb1 and Rvb2 are very similar and highly conserved members of the AAA+ (ATPase associated with diverse cellular activities) superfamily of ATPases, and they show homology to the bacterial RuvB DNA helicase. In addition to the biogenesis of box C/D and H/ACA snoRNPs, Rvb1/Rvb2-containing complexes also function in transcription activation and repression, cell transformation, DNA damage response, and DNA replication (Jha and Dutta, 2009). Rvb1 and Rvb2 form homo- or heterohexameric rings. In the R2TP complex, the Rvb1-Rvb2 hexamer tightly associates with the Tah1-Pih1 heterodimer through interactions with Pih1 (Boulon et al., 2008; Zhao et al., 2008). The correct molecular roles that Hsp90 and the R2TP complex play in H/ACA RNP assembly are unclear. In vitro pull-down and yeast two-hybrid assays show that Hsp90 directly interacts with Tah1 and Pih1, but in living cells Hsp90 shows no significant interaction with the R2TP complex. Since inhibition of Hsp90 activity prevents accumulation of Pih1, Hsp90 may provide stability for Pih1 and may promote its association with Tah1 and the Rvb1Rvb2 complex (Zhao et al., 2008). The R2TP chaperone complex is tethered to the maturing L7Ae RNPs by interactions with Rsa1 (Nufip in human) that binds to all members of the L7Ae RNP protein family, Nhp2, 15.5K, and Sbp2 (Boulon et al., 2008). In conclusion, biogenesis of eukaryotic H/ACA RNPs requires an unexpected number of trans-acting factors either specific for H/ACA RNPs (Naf1 and Shq1) or, more frequently, general assembly factors (Hsp90, R2TP complex, Nufip) that are involved in the biogenesis of various macromolecular complexes. A complete understanding of the biogenesis of eukaryotic H/ACA RNPs will apparently require much effort in the future. Targeting H/ACA scaRNPs into the Cajal Body Pseudouridylation of spliceosomal snRNAs is mediated by H/ ACA scaRNPs in the nucleoplasmic CBs (Darzacq et al., 2002). CBs are multifunctional dynamic structures enriched in factors involved in RNP assembly and RNA modification (Cioce and Lamond, 2005). The scaRNP-mediated pseudouridylation of snRNAs occurs after nuclear importation of the nascent snRNPs, which transit through CBs before accumulating in the interchromatin granules (Ja´dy et al., 2003). The H/ACA scaRNAs carry a short CB localization signal, the CAB box (ugAG), that is located in the apical loop of the 50 - and/or 30 -hairpin of the RNA (Richard et al., 2003; Theimer et al., 2007) (Figure 1B). Disruption of the CAB box of H/ACA scaRNAs results in a nucleolar accumulation of the mutant RNAs, and H/ACA snoRNAs can be targeted into the CB by adding a CAB box motif. Vertebrate H/ACA scaRNAs are frequently fused to box C/D RNAs that

Molecular Cell 37, March 12, 2010 ª2010 Elsevier Inc. 601

Molecular Cell

Review

Figure 4. The 30 -Hairpin of snR30/U17 snoRNA Forms a Complex Interaction with 18S rRNA Sequences The invariant rRNA (rm1 and rm2) and snoRNA (m1 and m2) sequences are shown in red and brown, respectively. The terminal stem-loop region interacts with putative rRNA processing factor(s) (PRPF). The box H/ACA core proteins are not shown.

function in the 20 -O-methylation of snRNAs (Darzacq et al., 2002; Ja´dy and Kiss, 2001). The composite H/ACA-C/D scaRNAs utilize the CAB box of the H/ACA domain as a CB targeting signal. Recently, a novel H/ACA scaRNP-specific protein, called WDR79 or TCAB1, has been identified (Tycowski et al., 2009; Venteicher et al., 2009). The human and Drosophila WDR79/ TCAB1 proteins accumulate in CBs, and they stably associate with H/ACA scaRNPs, including the human telomerase, in a CAB box-dependent manner. Association with WDR79/TCAB1 is required for CB localization of H/ACA scaRNPs. Interestingly, WDR79/TCAB1 also interacts with both human and fly box C/D scaRNPs, despite the fact that human box C/D scaRNAs lack recognizable CAB box motifs (Tycowski et al., 2009). The Drosophila box C/D scaRNAs carry a common CB localization sequence that seems to be an extended version of the vertebrate H/ACA CAB box. Recruitment of WDR79/TCAB1 to H/ACA scaRNAs, in addition to the CAB box, also requires the ACA box, indicating that WDR79/TCAB1 interacts with H/ACA core protein(s) (Richard et al., 2003; Tycowski et al., 2009; Venteicher et al., 2009). Understanding of the CB-specific localization of scaRNPs further emphasized that correct subcellular targeting of the assembled catalytically active H/ACA RNPs is an important step of the biogenesis of functional H/ACA RNPs.

602 Molecular Cell 37, March 12, 2010 ª2010 Elsevier Inc.

Other and New Functions for H/ACA RNPs Although the majority of box H/ACA RNAs function in rRNA and snRNA pseudouridylation, some H/ACA RNAs participate in pre-rRNA processing, telomere synthesis, and other unexpected processes. Processing of H/ACA RNAs into Small Regulatory RNAs MicroRNAs (miRNAs) are small 20–24 nt long regulatory RNAs which in collaboration with Argonaute (Ago) proteins function in posttranscriptional gene silencing through base pairing with target mRNAs (Chekulaeva and Filipowicz, 2009). Mature miRNAs are generated by the RNase III-type endoribonuclease Dicer from 60–80 nt long hairpin-shaped RNAs (pre-miRNAs) similar to the hairpin domains of H/ACA RNAs. It has been found that the 30 -hairpin of human ACA45 RNA, an H/ACA scaRNA directing pseudouridylation of the U2 snRNA, is processed into 20–25 nt long miRNA-like stable RNAs (Ender et al., 2008). The ACA45-derived small RNAs are associated with Ago, and they can function as genuine miRNAs targeting the endogenous mediator subunit CDC2L6 mRNA. Systematic analysis of small RNA libraries identified numerous miRNA-like RNAs derived from box H/ACA and C/D snoRNAs in vertebrates, Drosophila, plants, fission yeast, and the unicellular ancient eukaryote Giardia (Ender et al., 2008; Saraiya and Wang, 2008; Taft et al., 2009). Although the biological significance of these putative miRNAs is unclear, it is possible that processing of H/ACA and C/D snoRNA sequences is a widespread mechanism for generation of miRNAs. The snR30 H/ACA snoRNA Functions in 18S rRNA Processing The yeast snR30 (U17 in vertebrates) H/ACA snoRNA, contrary to its association with all H/ACA core proteins including the Cbf5 pseudouridine synthase, plays an essential role in the nucleolytic processing of 18S rRNA from the 35S pre-rRNA (Morrissey and Tollervey, 1993). It has been recently shown that the 30 terminal hairpin of snR30 carries an internal loop similar to the pseudouridylation pocket of modification guide RNAs that forms an unusual and very complex interaction with 18S rRNA sequences (Atzorn et al., 2004; Fayet-Lebaron et al., 2009) (Figure 4). Two conserved motifs, m1 and m2, located in the proximal (lower) part of the rRNA-recognition loop of snR30 base pair with short internal 18S sequences, rm1 and rm2, which are separated by a short stem loop. In addition to the sequences forming the snR30-18S interaction, processing of 18S rRNA also requires snoRNA elements located in the distal part of the 30 -hairpin of snR30. This region of snR30 is believed to bind putative pre-rRNA processing factors (PRPFs). While dissociation of H/ACA pseudouridylation RNPs from their substrate RNAs seems to be controlled by the Gar1 H/ACA protein and does not require additional trans-acting factors, the yeast snR30 is released from the maturing pre-rRNA by a specific RNA helicase, Rok1 (Bohnsack et al., 2008). Thus, similarly to the classical pseudouridylation-guide RNAs, the snR30 snoRNA also functions as a guide RNA that transiently base pairs with pre-rRNA sequences to target essential processing factors to the 18S rRNA undergoing nucleolytic maturation. Telomerase Is a CB-Specific Box H/ACA RNP Telomerase is an H/ACA RNP enzyme responsible for addition of telomeric DNA repeats to the termini of eukaryotic linear

Molecular Cell

Review

Figure 5. Synthesis of Human Telomeric DNA by Telomerase Schematic structure of the human telomerase RNA (hTR) with the functionally important sequence elements is shown. The template sequence of hTR (purple) recognizes the terminal nucleotides of the telomeric G-rich strand and dictates its elongation by the associated telomerase reverse transcriptase (hTERT). The nascent telomeric DNA is in green. The CAB box (blue) and 30 end-processing signal (orange) of hTR are indicated.

chromosomes (Collins, 2006). Telomerase activity counteracts telomere attrition and is essential for the viability of most proliferating cells, including cancer cells and stem cells. The human telomerase RNA (hTR) is composed of a 50 -terminal pseudoknot domain that provides the telomeric template sequence for the associated telomerase reverse transcriptase (hTERT) and a 30 -terminal box H/ACA domain that is essential for the accumulation, enzymatic activity, and correct localization of telomerase RNP (Figure 5) (Chen et al., 2000, 2002; Fu and Collins, 2003; Mitchell et al., 1999; Mitchell and Collins, 2000; Theimer et al., 2007). The apical stem-loop region of the 30 -hairpin of hTR encompasses a CB localization signal (CAB box) and another structurally overlapping, telomerase-specific element that is essential for efficient 30 end processing of hTR (Ja´dy et al., 2004; Theimer et al., 2007). The H/ACA scaRNA domain of hTR interacts with the four H/ACA core proteins and the scaRNPspecific WDR79/TCAB1 protein (Cohen et al., 2007; Tycowski et al., 2009; Venteicher et al., 2009). The human telomerase scaRNP accumulates in CBs in a CAB box- and WDR79/TCAB1-dependent manner (Ja´dy et al., 2004; Venteicher et al., 2009; Zhu et al., 2004). Mutant TRs lacking functional CAB box motifs, although incorporated into catalytically active telomerase particles, are impaired in in vivo telomere elongation (Cristofari et al., 2007). Likewise, depletion of

WDR79/TCAB1 results in telomerase mislocalization and inefficient telomere synthesis but has no effect on the accumulation of catalytically competent telomerase (Venteicher et al., 2009). Therefore, localization of telomerase to CBs is important for telomerase function, rather than for the assembly of active telomerase. Besides accumulating in CBs, HeLa cell telomerase also concentrates at a few (usually one to three) telomeres during S phase when telomere synthesis is known to occur (Ja´dy et al., 2006; Tomlinson et al., 2006). This might indicate that human telomerase elongates only a subset of telomeres within one cell cycle (Teixeira et al., 2004). Intriguingly, more than 25% of HeLa telomeres accumulating telomerase colocalize with CBs (Ja´dy et al., 2006). CBs are highly mobile subnuclear organelles that transiently associate with actively transcribed chromosome territories, suggesting that they also function in the intranuclear transport and sorting of nuclear factors (Cioce and Lamond, 2005). In vivo imaging demonstrated that CBs moving in the interchromatin space of HeLa S phase cells transiently associate with telomeres with the frequency of about two interactions per hour (Ja´dy et al., 2006). This means that CBs can visit five to seven telomeres during the entire S phase and therefore CBs not only store telomerase but they may also deliver it to telomeres in a cell-cycle-regulated fashion. Dyskeratosis congenita (DC) is an inherited disorder characterized by heterogeneous pathological features, including bone marrow failure, premature aging, and increased susceptibility to cancer (Kirwan and Dokal, 2009). DC is caused by mutations in genes encoding the human telomerase components dyskerin, hTR, hTERT, Nop10, Nhp2, or the telomere protecting protein Tin2. Accordingly, the presence of abnormally short telomeres is a common molecular feature and most probably the major underlying cause of DC. The most frequent and severe X-linked form of DC is caused by missense mutations in the dyskerin gene. Determination of the crystal structure of archaeal Cbf5 revealed that DC mutations, while distantly located on the primary structure, are clustered in the PUA three-dimensional domain of dyskerin that is responsible for binding the ACA box motif of H/ACA RNAs (Rashid et al., 2006). More recently, it has been shown that the PUA domain of dyskerin also provides the binding surface for the H/ACA assembly factor Shq1 and that DC mutations can influence the interaction of dyskerin and Shq1 (Grozdanov et al., 2009a). These observations, together with the fact that DC cells show reduced H/ACA RNPs accumulation, suggest that weakened interaction of dyskerin with the H/ACA domain of hTR or with the H/ACA assembly factor Shq1 may be the molecular cause of X-linked DC. In mouse and Drosophila models, DC mutations have been shown to associate with ribosomal defects, suggesting that impaired H/ACA snoRNP function could also contribute to the development of DC symptoms (Kirwan and Dokal, 2009). Conclusions Understanding the structural organization of archaeal H/ACA RNPs revealed the major principles of the enzymatic mechanism of this unique class of pseudouridine synthases. The H/ACA pseudouridylation RNPs and the stand-alone pseudouridine synthases share strong structural and mechanistic similarities, but in contrast to the protein enzymes, the H/ACA RNPs possess

Molecular Cell 37, March 12, 2010 ª2010 Elsevier Inc. 603

Molecular Cell

Review the capacity to recruit substrate RNAs with diverse nucleotide and local structural composition and to convert them into a catalytic-competent conformation. The biogenesis of eukaryotic H/ACA RNPs is assisted by a cohort of evolutionarily conserved RNP assembly factors, and the assembly of H/ACA RNPs is accompanied by a complex intranuclear trafficking before the mature RNPs accumulate in the nucleolus or in the CB. The complexity of H/ACA RNP trafficking has been further emphasized by the finding that human telomerase accumulates at telomeres in a cell-cycle-dependent manner. ACKNOWLEDGMENTS This work was supported by grants from la Fondation pour la Recherche Me´dicale and l’Agence Nationale de Recherche sur le Sida. We apologize to our colleagues whose work could not be cited due to size constraints.

Cohen, S.B., Graham, M.E., Lovrecz, G.O., Bache, N., Robinson, P.J., and Reddel, R.R. (2007). Protein composition of catalytically active human telomerase from immortal cells. Science 315, 1850–1853. Collins, K. (2006). The biogenesis and regulation of telomerase holoenzymes. Nat. Rev. Mol. Cell Biol. 7, 484–494. Cristofari, G., Adolf, E., Reichenbach, P., Sikora, K., Terns, R.M., Terns, M.P., and Lingner, J. (2007). Human telomerase RNA accumulation in Cajal bodies facilitates telomerase recruitment to telomeres and telomere elongation. Mol. Cell 27, 882–889. Darzacq, X., Ja´dy, B.E., Verheggen, C., Kiss, A.M., Bertrand, E., and Kiss, T. (2002). Cajal body-specific small nuclear RNAs: a novel class of 20 -O-methylation and pseudouridylation guide RNAs. EMBO J. 21, 2746–2756. Darzacq, X., Kittur, N., Roy, S., Shav-Tal, Y., Singer, R.H., and Meier, U.T. (2006). Stepwise RNP assembly at the site of H/ACA RNA transcription in human cells. J. Cell Biol. 173, 207–218. Dennis, P.P., and Omer, A. (2005). Small non-coding RNAs in Archaea. Curr. Opin. Microbiol. 8, 685–694.

REFERENCES

Dez, C., Noaillac-Depeyre, J., Caizergues-Ferrer, M., and Henry, Y. (2002). Naf1p, an essential nucleoplasmic factor specifically required for accumulation of box H/ACA small nucleolar RNPs. Mol. Cell. Biol. 22, 7053–7065.

Atzorn, V., Fragapane, P., and Kiss, T. (2004). U17/snR30 is a ubiquitous snoRNA with two conserved sequence motifs essential for 18S rRNA production. Mol. Cell. Biol. 24, 1769–1778.

Duan, J., Li, L., Lu, J., Wang, W., and Ye, K. (2009). Structural mechanism of substrate RNA recruitment in H/ACA RNA-guided pseudouridine synthase. Mol. Cell 34, 427–439.

Baker, D.L., Youssef, O.A., Chastkofsky, M.I., Dy, D.A., Terns, R.M., and Terns, M.P. (2005). RNA-guided RNA modification: functional organization of the archaeal H/ACA RNP. Genes Dev. 19, 1238–1248.

Ender, C., Krek, A., Friedlander, M.R., Beitzinger, M., Weinmann, L., Chen, W., Pfeffer, S., Rajewsky, N., and Meister, G. (2008). A human snoRNA with microRNA-like functions. Mol. Cell 32, 519–528.

Balakin, A.G., Smith, L., and Fournier, M.J. (1996). The RNA world of the nucleolus: two major families of small RNAs defined by different box elements with related functions. Cell 86, 823–834.

Fatica, A., Dlakic, M., and Tollervey, D. (2002). Naf1 p is a box H/ACA snoRNP assembly factor. RNA 8, 1502–1514.

Ballarino, M., Morlando, M., Pagano, F., Fatica, A., and Bozzoni, I. (2005). The cotranscriptional assembly of snoRNPs controls the biosynthesis of H/ACA snoRNAs in Saccharomyces cerevisiae. Mol. Cell. Biol. 25, 5396–5403. Bally, M., Hughes, J., and Cesareni, G. (1988). SnR30: a new, essential small nuclear RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 16, 5291–5303. Bohnsack, M.T., Kos, M., and Tollervey, D. (2008). Quantitative analysis of snoRNA association with pre-ribosomes and release of snR30 by Rok1 helicase. EMBO Rep. 9, 1230–1236. Boulon, S., Verheggen, C., Ja´dy, B.E., Girard, C., Pescia, C., Paul, C., Ospina, J.K., Kiss, T., Matera, A.G., Bordonne, R., et al. (2004). PHAX and CRM1 are required sequentially to transport U3 snoRNA to nucleoli. Mol. Cell 16, 777–787. Boulon, S., Marmier-Gourrier, N., Pradet-Balade, B., Wurth, L., Verheggen, C., Ja´dy, B.E., Rothe, B., Pescia, C., Robert, M.C., Kiss, T., et al. (2008). The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery. J. Cell Biol. 180, 579–595. Charette, M., and Gray, M.W. (2000). Pseudouridine in RNA: what, where, how, and why. IUBMB Life 49, 341–351. Charpentier, B., Muller, S., and Branlant, C. (2005). Reconstitution of archaeal H/ACA small ribonucleoprotein complexes active in pseudouridylation. Nucleic Acids Res. 33, 3133–3144. Chekulaeva, M., and Filipowicz, W. (2009). Mechanisms of miRNA-mediated post-transcriptional regulation in animal cells. Curr. Opin. Cell Biol. 21, 452–460. Chen, J.L., Blasco, M.A., and Greider, C.W. (2000). Secondary structure of vertebrate telomerase RNA. Cell 100, 503–514. Chen, J.L., Opperman, K.K., and Greider, C.W. (2002). A critical stem-loop structure in the CR4-CR5 domain of mammalian telomerase RNA. Nucleic Acids Res. 30, 592–597. Cioce, M., and Lamond, A.I. (2005). Cajal bodies: a long history of discovery. Annu. Rev. Cell Dev. Biol. 21, 105–131.

604 Molecular Cell 37, March 12, 2010 ª2010 Elsevier Inc.

Fayet-Lebaron, E., Atzorn, V., Henry, Y., and Kiss, T. (2009). 18S rRNA processing requires base pairings of snR30 H/ACA snoRNA to eukaryotespecific 18S sequences. EMBO J. 28, 1260–1270. Fu, D., and Collins, K. (2003). Distinct biogenesis pathways for human telomerase RNA and H/ACA small nucleolar RNAs. Mol. Cell 11, 1361–1372. Ganot, P., Bortolin, M.L., and Kiss, T. (1997a). Site-specific pseudouridine formation in preribosomal RNA is guided by small nucleolar RNAs. Cell 89, 799–809. Ganot, P., Caizergues-Ferrer, M., and Kiss, T. (1997b). The family of box ACA small nucleolar RNAs is defined by an evolutionarily conserved secondary structure and ubiquitous sequence elements essential for RNA accumulation. Genes Dev. 11, 941–956. Godin, K.S., Walbott, H., Leulliot, N., van Tilbeurgh, H., and Varani, G. (2009). The box H/ACA snoRNP assembly factor Shq1p is a chaperone protein homologous to Hsp90 cochaperones that binds to the Cbf5p enzyme. J. Mol. Biol. 390, 231–244. Grozdanov, P.N., Fernandez-Fuentes, N., Fiser, A., and Meier, U.T. (2009a). Pathogenic Nap57 mutations decrease ribonucleoprotein assembly in dyskeratosis congenita. Hum. Mol. Genet. 18, 4546–4551. Grozdanov, P.N., Roy, S., Kittur, N., and Meier, U.T. (2009b). SHQ1 is required prior to NAF1 for assembly of H/ACA small nucleolar and telomerase RNPs. RNA 15, 1188–1197. Hamma, T., and Ferre-D’Amare, A.R. (2004). Structure of protein L7Ae bound to a K-turn derived from an archaeal box H/ACA sRNA at 1.8 A˚ resolution. Structure 12, 893–903. Hamma, T., and Ferre-D’Amare, A.R. (2006). Pseudouridine synthases. Chem. Biol. 13, 1125–1135. Hamma, T., Reichow, S.L., Varani, G., and Ferre-D’Amare, A.R. (2005). The Cbf5-Nop10 complex is a molecular bracket that organizes box H/ACA RNPs. Nat. Struct. Mol. Biol. 12, 1101–1107. Henras, A.K., Capeyrou, R., Henry, Y., and Caizergues-Ferrer, M. (2004). Cbf5p, the putative pseudouridine synthase of H/ACA-type snoRNPs, can form a complex with Gar1p and Nop10p in absence of Nhp2p and box H/ACA snoRNAs. RNA 10, 1704–1712.

Molecular Cell

Review Hoang, C., and Ferre-D’Amare, A.R. (2001). Cocrystal structure of a tRNA Psi55 pseudouridine synthase: nucleotide flipping by an RNA-modifying enzyme. Cell 107, 929–939.

Mitchell, J.R., and Collins, K. (2000). Human telomerase activation requires two independent interactions between telomerase RNA and telomerase reverse transcriptase. Mol. Cell 6, 361–371.

Ja´dy, B.E., and Kiss, T. (2001). A small nucleolar guide RNA functions both in 20 -O-ribose methylation and pseudouridylation of the U5 spliceosomal RNA. EMBO J. 20, 541–551.

Mitchell, J.R., Cheng, J., and Collins, K. (1999). A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 30 end. Mol. Cell. Biol. 19, 567–576.

Ja´dy, B.E., Darzacq, X., Tucker, K.E., Matera, A.G., Bertrand, E., and Kiss, T. (2003). Modification of Sm small nuclear RNAs occurs in the nucleoplasmic Cajal bodies following import from the cytoplasm. EMBO J. 22, 1878–1888.

Morrissey, J.P., and Tollervey, D. (1993). Yeast snR30 is a small nucleolar RNA required for 18S rRNA synthesis. Mol. Cell. Biol. 13, 2469–2477.

Ja´dy, B.E., Bertrand, E., and Kiss, T. (2004). Human telomerase RNA and box H/ACA scaRNAs share a common Cajal body-specific localization signal. J. Cell Biol. 164, 647–652.

Normand, C., Capeyrou, R., Quevillon-Cheruel, S., Mougin, A., Henry, Y., and Caizergues-Ferrer, M. (2006). Analysis of the binding of the N-terminal conserved domain of yeast Cbf5p to a box H/ACA snoRNA. RNA 12, 1868– 1882.

Ja´dy, B.E., Richard, P., Bertrand, E., and Kiss, T. (2006). Cell cycle-dependent recruitment of telomerase RNA and Cajal bodies to human telomeres. Mol. Biol. Cell 17, 944–954.

Omer, A.D., Lowe, T.M., Russell, A.G., Ebhardt, H., Eddy, S.R., and Dennis, P.P. (2000). Homologs of small nucleolar RNAs in Archaea. Science 288, 517–522.

Jha, S., and Dutta, A. (2009). RVB1/RVB2: running rings around molecular biology. Mol. Cell 34, 521–533. Jin, H., Loria, J.P., and Moore, P.B. (2007). Solution structure of an rRNA substrate bound to the pseudouridylation pocket of a box H/ACA snoRNA. Mol. Cell 26, 205–215. Kirwan, M., and Dokal, I. (2009). Dyskeratosis congenita, stem cells and telomeres. Biochim. Biophys. Acta 1792, 371–379.

Pan, H., Agarwalla, S., Moustakas, D.T., Finer-Moore, J., and Stroud, R.M. (2003). Structure of tRNA pseudouridine synthase TruB and its RNA complex: RNA recognition through a combination of rigid docking and induced fit. Proc. Natl. Acad. Sci. USA 100, 12648–12653. Pearl, L.H., and Prodromou, C. (2006). Structure and mechanism of the Hsp90 molecular chaperone machinery. Annu. Rev. Biochem. 75, 271–294.

Kiss, T., Fayet, E., Ja´dy, B.E., Richard, P., and Weber, M. (2006). Biogenesis and intranuclear trafficking of human box C/D and H/ACA RNPs. Cold Spring Harb. Symp. Quant. Biol. 71, 407–417.

Rashid, R., Liang, B., Baker, D.L., Youssef, O.A., He, Y., Phipps, K., Terns, R.M., Terns, M.P., and Li, H. (2006). Crystal structure of a Cbf5-Nop10-Gar1 complex and implications in RNA-guided pseudouridylation and dyskeratosis congenita. Mol. Cell 21, 249–260.

Kittur, N., Darzacq, X., Roy, S., Singer, R.H., and Meier, U.T. (2006). Dynamic association and localization of human H/ACA RNP proteins. RNA 12, 2057– 2062.

Richard, P., Darzacq, X., Bertrand, E., Ja´dy, B.E., Verheggen, C., and Kiss, T. (2003). A common sequence motif determines the Cajal body-specific localisation of box H/ACA scaRNAs. EMBO J. 22, 4283–4293.

Koonin, E.V. (1996). Pseudouridine synthases: four families of enzymes containing a putative uridine-binding motif also conserved in dUTPases and dCTP deaminases. Nucleic Acids Res. 24, 2411–2415.

Richard, P., Kiss, A.M., Darzacq, X., and Kiss, T. (2006). Cotranscriptional recognition of human intronic box H/ACA snoRNAs occurs in a splicingindependent manner. Mol. Cell. Biol. 26, 630–642.

Kuhn, J.F., Tran, E.J., and Maxwell, E.S. (2002). Archaeal ribosomal protein L7 is a functional homolog of the eukaryotic 15.5kD/Snu13p snoRNP core protein. Nucleic Acids Res. 30, 931–941.

Roovers, M., Hale, C., Tricot, C., Terns, M.P., Terns, R.M., Grosjean, H., and Droogmans, L. (2006). Formation of the conserved pseudouridine at position 55 in archaeal tRNA. Nucleic Acids Res. 34, 4293–4301.

Lafontaine, D.L., Bousquet-Antonelli, C., Henry, Y., Caizergues-Ferrer, M., and Tollervey, D. (1998). The box H + ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase. Genes Dev. 12, 527–537.

Rozhdestvensky, T.S., Tang, T.H., Tchirkova, I.V., Brosius, J., Bachellerie, J.P., and Huttenhofer, A. (2003). Binding of L7Ae protein to the K-turn of archaeal snoRNAs: a shared RNA binding motif for C/D and H/ACA box snoRNAs in Archaea. Nucleic Acids Res. 31, 869–877.

Leulliot, N., Godin, K.S., Hoareau-Aveilla, C., Quevillon-Cheruel, S., Varani, G., Henry, Y., and Van Tilbeurgh, H. (2007). The box H/ACA RNP assembly factor Naf1p contains a domain homologous to Gar1p mediating its interaction with Cbf5p. J. Mol. Biol. 371, 1338–1353. Li, L., and Ye, K. (2006). Crystal structure of an H/ACA box ribonucleoprotein particle. Nature 443, 302–307. Liang, B., Xue, S., Terns, R.M., Terns, M.P., and Li, H. (2007). Substrate RNA positioning in the archaeal H/ACA ribonucleoprotein complex. Nat. Struct. Mol. Biol. 14, 1189–1195. Liang, B., Zhou, J., Kahen, E., Terns, R.M., Terns, M.P., and Li, H. (2009). Structure of a functional ribonucleoprotein pseudouridine synthase bound to a substrate RNA. Nat. Struct. Mol. Biol. 16, 740–746. Luo, Y., and Li, S. (2007). Genome-wide analyses of retrogenes derived from the human box H/ACA snoRNAs. Nucleic Acids Res. 35, 559–571. Manival, X., Charron, C., Fourmann, J.B., Godard, F., Charpentier, B., and Branlant, C. (2006). Crystal structure determination and site-directed mutagenesis of the Pyrococcus abyssi aCBF5-aNOP10 complex reveal crucial roles of the C-terminal domains of both proteins in H/ACA sRNP activity. Nucleic Acids Res. 34, 826–839. Matera, A.G., Terns, R.M., and Terns, M.P. (2007). Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs. Nat. Rev. Mol. Cell Biol. 8, 209–220. Meier, U.T. (2005). The many facets of H/ACA ribonucleoproteins. Chromosoma 114, 1–14.

Saraiya, A.A., and Wang, C.C. (2008). snoRNA, a novel precursor of microRNA in Giardia lamblia. PLoS Pathog. 4, e1000224. 10.1371/journal.ppat.1000224. Singh, M., Gonzales, F.A., Cascio, D., Heckmann, N., Chanfreau, G., and Feigon, J. (2009). Structure and functional studies of the CS domain of the essential H/ACA ribonucleoparticle assembly protein SHQ1. J. Biol. Chem. 284, 1906–1916. Taft, R.J., Glazov, E.A., Lassmann, T., Hayashizaki, Y., Carninci, P., and Mattick, J.S. (2009). Small RNAs derived from snoRNAs. RNA 15, 1233–1240. Teixeira, M.T., Arneric, M., Sperisen, P., and Lingner, J. (2004). Telomere length homeostasis is achieved via a switch between telomerase-extendible and -nonextendible states. Cell 117, 323–335. Terns, M.P., and Terns, R. (2006). Noncoding RNAs of the H/ACA family. Cold Spring Harb. Symp. Quant. Biol. LXXI, 395–405. Theimer, C.A., Ja´dy, B.E., Chim, N., Richard, P., Breece, K.E., Kiss, T., and Feigon, J. (2007). Structural and functional characterization of human telomerase RNA processing and cajal body localization signals. Mol. Cell 27, 869–881. Tollervey, D. (1987). A yeast small nuclear RNA is required for normal processing of pre-ribosomal RNA. EMBO J. 6, 4169–4175. Tomlinson, R.L., Ziegler, T.D., Supakorndej, T., Terns, R.M., and Terns, M.P. (2006). Cell cycle-regulated trafficking of human telomerase to telomeres. Mol. Biol. Cell 17, 955–965.

Molecular Cell 37, March 12, 2010 ª2010 Elsevier Inc. 605

Molecular Cell

Review Tycowski, K.T., Shu, M.D., Kukoyi, A., and Steitz, J.A. (2009). A conserved WD40 protein binds the Cajal body localization signal of scaRNP particles. Mol. Cell 34, 47–57.

Yang, P.K., Rotondo, G., Porras, T., Legrain, P., and Chanfreau, G. (2002). The Shq1p.Naf1p complex is required for box H/ACA small nucleolar ribonucleoprotein particle biogenesis. J. Biol. Chem. 277, 45235–45242.

Venteicher, A.S., Meng, Z., Mason, P.J., Veenstra, T.D., and Artandi, S.E. (2008). Identification of ATPases pontin and reptin as telomerase components essential for holoenzyme assembly. Cell 132, 945–957.

Yang, P.K., Hoareau, C., Froment, C., Monsarrat, B., Henry, Y., and Chanfreau, G. (2005). Cotranscriptional recruitment of the pseudouridylsynthetase Cbf5p and of the RNA binding protein Naf1p during H/ACA snoRNP assembly. Mol. Cell. Biol. 25, 3295–3304.

Venteicher, A.S., Abreu, E.B., Meng, Z., McCann, K.E., Terns, R.M., Veenstra, T.D., Terns, M.P., and Artandi, S.E. (2009). A human telomerase holoenzyme protein required for Cajal body localization and telomere synthesis. Science 323, 644–648. Wang, C., and Meier, U.T. (2004). Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins. EMBO J. 23, 1857–1867. Watkins, N.J., Gottschalk, A., Neubauer, G., Kastner, B., Fabrizio, P., Mann, M., and Lu¨hrmann, R. (1998). Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure. RNA 4, 1549–1568.

Yu, Y.T., Terns, R.M., and Terns, M.P. (2005). Mechanisms and functions of RNA-guided RNA modification (New York: Springer-Verlag). Zebarjadian, Y., King, T., Fournier, M.J., Clarke, L., and Carbon, J. (1999). Point mutations in yeast CBF5 can abolish in vivo pseudouridylation of rRNA. Mol. Cell. Biol. 19, 7461–7472. Zhao, R., Davey, M., Hsu, Y.C., Kaplanek, P., Tong, A., Parsons, A.B., Krogan, N., Cagney, G., Mai, D., Greenblatt, J., et al. (2005). Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone. Cell 120, 715–727.

Weber, M.J. (2006). Mammalian small nucleolar RNAs are mobile genetic elements. PLoS Genet. 2, e205. 10.1371/journal.pgen.0020205.

Zhao, R., Kakihara, Y., Gribun, A., Huen, J., Yang, G., Khanna, M., Costanzo, M., Brost, R.L., Boone, C., Hughes, T.R., et al. (2008). Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation. J. Cell Biol. 180, 563–578.

Wu, H., and Feigon, J. (2007). H/ACA small nucleolar RNA pseudouridylation pockets bind substrate RNA to form three-way junctions that position the target U for modification. Proc. Natl. Acad. Sci. USA 104, 6655–6660.

Zhu, Y., Tomlinson, R.L., Lukowiak, A.A., Terns, R.M., and Terns, M.P. (2004). Telomerase RNA accumulates in Cajal bodies in human cancer cells. Mol. Biol. Cell 15, 81–90.

606 Molecular Cell 37, March 12, 2010 ª2010 Elsevier Inc.