Architecture of human telomerase RNAQi Zhang, Nak-Kyoon Kim, and Juli Feigon1

Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-1569

Edited by Neal F. Lue, Weill Cornell Medical College, New York, NY, and accepted by the Editorial Board July 22, 2011 (received for review June 8, 2011)

Telomerase is a unique reverse transcriptase that catalyzes the addition of telomere DNA repeats onto the 3′ ends of linear chromosomesand plays a critical role in maintaining genome stability. Unlike other reverse transcriptases, telomerase is unique in that it is a ribonu-cleoprotein complex, where the RNA component [telomerase RNA (TR)] not only provides the template for the synthesis of telomere DNArepeats but also plays essential roles in catalysis, accumulation, TR 3′-end processing, localization, and holoenzyme assembly. Biochemicalstudies have identified TR elements essential for catalysis that share remarkably conserved secondary structures across different species aswell as species-specific domains for other functions, paving the way for high-resolution structure determination of TRs. Over the pastdecade, structures of key elements from the core, conserved regions 4 and 5, and small Cajal body specific RNA domains of human TR haveemerged, providing significant insights into the roles of these RNA elements in telomerase function. Structures of all helical elements ofthe core domain have been recently reported, providing the basis for a high-resolution model of the complete core domain. We reviewthis progress to determine the overall architecture of human telomerase RNA.

Box H/ACA RNA | NMR | pseudoknot | telomerase reverse transcriptase

Telomerase is a large, multisubunitribonucleoprotein (RNP) thatreplicates the 3′ end of linearchromosomes by processive syn-

thesis of telomere DNA repeats. Telo-meres, the physical ends of linearchromosomes, generally comprise dsDNAwith a short repeating species-specific se-quence ending in a 3′ single-strandedoverhang of variable length plus associatedtelomere binding proteins, called shelterinin humans (1, 2). Telomeres protect theintegrity of linear chromosomes by allow-ing the cellular DNA repair machineryto distinguish them from double-strandbreaks, thus playing critical roles in main-taining genome stability in eukaryotes (1,2). Shortening of telomeres below a criti-cal length because of inherent incompletereplication of DNA ends ultimately leadsto telomere fusions and cell senescence(3–6). The 3′ ends of telomeres are repli-cated by telomerase, a unique reversetranscriptase discovered almost threedecades ago (7), which catalyzes the ad-dition of telomere DNA repeats onto theends of linear chromosomes using anembedded RNA as the template (8, 9).Although telomerase has a low orundetectable level of activity in most so-matic cells, it is active in some germline,epithelial, and hematopoietic cells, and itis highly active in the majority (∼90%) ofcancer cell lines (10–12). Telomerase de-ficiency because of mutations in humantelomerase RNA (hTR) has also beenlinked to several inherited human dis-eases, such as dyskeratosis congenita,aplastic anemia, myelodysplasia, and idio-pathic pulmonary fibrosis (13–26).The telomerase holoenzyme includes

a unique reverse transcriptase [telomerasereverse transcriptase (TERT)], an essentialRNA (TR), and several species-specificproteins required for proper function invivo (27–29). The protein TERT is highly

conserved across different species, and itusually contains four major functionaldomains: the TERT N-terminal domain(TEN), the TERT RNA binding domain(TRBD), the reverse transcriptase domain(RT), and the C-terminal extension (27,30–32). The TEN domain interacts withthe ss telomere DNA repeats, the TRBDdomain binds multiple sites of TR, and theRT and C-terminal extension domainsbind the RNA/DNA hybrid and catalyzethe addition of DNA repeats onto the 3′end (27, 31–34). Although no structures ofTERT with all four domains have yet beenreported for any species, structures of theTEN and TRBD domains from Tetrahy-mena thermophila telomerase (33, 35) andthe full-length Tribolium castaneum TERTthat lacks the TEN domain have been re-ported (36, 37). These high-resolutionstructures have significantly advanced ourunderstanding of how TERT catalyzes thereverse transcription of telomere DNAand how TERT could potentially interactwith the ss telomere DNA and the tem-plate RNA/DNA hybrids. Excellent re-views can be found elsewhere that describethe current state of knowledge about thestructure and function of TERT (31, 32).Although the essential templating

function of TR was discovered more than20 y ago (38), the TR contains more thana template. To date, the TR sequences of28 ciliates, 43 vertebrates, and 25 yeastshave been determined (39–41). In contrastto the relatively conserved TERT, TRsdiffer greatly not only in sequence but alsoin length, ranging from 147 to 205 nt inciliates (38, 42–45), from 312 to 559 ntin vertebrates (46, 47), and from 779 to>2,030 nt in yeasts (40, 41). Despite sig-nificant challenges in identifying commonfunctional TR elements because of suchdivergence, phylogenetic and mutationalstudies have revealed a conserved sec-ondary structure found in common among

TRs across different species, which in-cludes a large loop containing the tem-plate, a 5′ template boundary element,a pseudoknot, a loop-closing helix, anda stem terminus element (STE) (9, 15, 46,48). The conservation of secondary struc-tures rather than sequences suggests a rolefor these RNA structural motifs in telo-merase function, and indeed, these regionsof TR are essential for synthesis of telo-mere repeats (49–55). Other regions ofTR are involved in species-specific roles intelomerase biogenesis, RNA processing,localization, and accumulation (56–61).A conserved secondary structure for

vertebrate TRs was first determined on thebasis of 35 sequences (46), with four pro-posed conserved structural domains: (i)the pseudoknot, which includes the tem-plate, and (ii) the conserved regions 4 and5 (CR4/CR5), which together comprisethe catalytic core of the TR; (iii) the boxH/ACA; and (iv) the CR7. An updatedsecondary structure of hTR (47, 62) isshown in Fig. 1 with known associatedholoenzyme proteins. All of the secondarystructure elements found in commonamong TRs across different species are inthe 5′ region of hTR, where the STE is the

This paper results from the Arthur M. Sackler Colloquiumof the National Academy of Sciences, “Telomerase andRetrotransposons: Reverse Transcriptases That Shaped Ge-nomes” held September 29–30, 2010, at the Arnold andMabel Beckman Center of the National Academies of Sci-ences and Engineering in Irvine, CA. The complete pro-gram and audio files of most presentations are availableon the NAS Web site at www.nasonline.org/telomerase_and_retrotransposons.

Author contributions: Q.Z., N.-K.K., and J.F. wrote thepaper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. N.F.L. is a guesteditor invited by the Editorial Board.1To whom correspondence should be addressed. E-mail:feigon@mbi.ucla.edu.

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CR4/CR5 domain (nucleotides ∼243 to∼326), and the remaining motifs comprisethe core domain, also known as the tem-plate/pseudoknot domain or the pseu-doknot/core domain (nucleotide 33–191)(46, 47, 63). These two highly conservedstructural domains independently bindTERT (50, 64) and are the only requiredhTR elements for in vitro reconstitutionof catalytically active telomerase withhTERT (49, 50). The 3′ end of hTR hasbeen identified as an H/ACA small Cajalbody (CB) -specific RNA (scaRNA; nu-cleotides ∼211 to ∼237 and ∼334 to 451,the upper boundary of the 5′ hairpin hasnot been determined) (59, 62, 65) (Fig. 1),and it plays essential roles in biogenesisand regulation of telomerase holoenzymein vivo, including accumulation, 3′-endprocessing, and localization of hTR (58–60, 66). The hTR scaRNA binds two setsof the four evolutionary conserved H/ACARNP proteins (dyskerin, Gar1, Nop10, andNhp2) to form an H/ACA RNP (Fig. 1)(67). Structures of single hairpin H/ACARNPs from archaea have provided insightinto the likely placement of proteins oneach hairpin of the hTR scaRNA domain(66, 68–71). The hTR scaRNA also con-tains a conserved Cajal body localizationelement (CAB box) (59) in its 3′ terminalhairpin loop in the region identified as theCR7 domain, identifying it as a scaRNP.The protein telomerase Cajal body protein1 (TCAB1)/WD-repeat domain 79(WDR79) that binds the CAB box anddrives localization of hTR into Cajal bodywas recently identified (72, 73).To date, there are no crystal structures of

telomerase RNA or telomerase protein–RNA complexes; however, over the lastseveral years, structures of several keyhTR elements have been determined byNMR spectroscopy (52, 54, 55, 60, 74–76).This review summarizes what NMRstructural and dynamics studies, combinedwith biochemical and mutational analysis,have revealed about the functional roles ofhTR domains and the overall architectureof hTR.

Core Domain of hTRThe core domain has been the main focusof biochemical and biophysical studies ofhTR, because it contains most of theconserved nucleotides and most of thedisease-linked mutations (21–24, 26, 48,62). It is the largest functional RNA do-main at the 5′ end of hTR. Biochemicalcharacterizations have identified nucleo-tides between residues 33 and 191 as theminimal hTR core domain required fortelomerase catalytic activity in vitro (63).This region can be further divided intothree major segments, a large loop con-taining the template, the P1 helix as theloop-closing helix, and a full-length P2/P3pseudoknot (Fig. 2A). Within the P2/P3

pseudoknot, all four helices (P2a.1, P2a,P2b, and P3) have been shown to be re-quired for telomerase activity, and disease-linked mutations disrupting these helicalstructures severely impair telomerase ca-talysis (63). Between these helical regionsare internal loop regions. Except for loopJ2b/3 and the 3′ portion of loop J2a/3,which are critical for the formation of P2b-P3 pseudoknot, most of these loop regionsare not conserved, and their nucleotideidentities are not essential for telomeraseactivity. The P2b-P3 pseudoknot is linkedto the P1 helix through three nucleotidesat its 3′ end and to the P2a helix throughan asymmetric bulge J2a/b at its 5′ end(Fig. 2A). On the other side of the P2ahelix are P2a.1 and the adjacent internalloop J2a.1, which are a mammalian-spe-cific extension to P2a helix (Fig. 2A). The5′ end of the P2a.1 helix and 3′ end of theP1 helix are linked by the large template-containing loop. These secondary struc-ture features suggest a unique and overallcompact architecture adopted by the hTRcore domain, because the ends of the ∼43-

bp P2/P3 pseudoknot are constrainedby the intervening 24-nt template-containing loop.

Structure and Dynamics of the P2b-P3 Pseu-doknot. The P2b-P3 pseudoknot containsalmost all of the highly conserved nucleo-tides within the hTR core domain (46).Early studies of minimal pseudoknotscontaining these conserved nucleotidesshowed that the pseudoknot unfolds toa P2b hairpin containing an unusual run ofU-U and U-C base pair (77) and thata two-base mutation in the P3 found insome patients with dyskeratosis congenita(13) destabilizes the pseudoknot. Al-though a conformational switch betweenthe completely folded pseudoknot and thispartially folded hairpin was proposed asa molecular switch for template trans-location (77, 78), both the solution struc-ture of the minimal pseudoknot (discussedbelow) and mutational analysis suggestthat a completely folded pseudoknot isrequired for optimal activity of telomerase(52, 79). The solution structure of theminimal hTR pseudoknot is a compact H-type pseudoknot with extensive tertiaryinteractions between the loop and stemnucleotides, and it revealed an essentialtriple helix in the pseudoknot (52). Thefunctional importance of the tertiary in-teractions, described below, was shown bycomparison of thermodynamic stabilitiesof pseudoknots with mutations and com-pensatory mutations and the effects ofthe same nucleotide substitutions in-corporated into the full-length TR on te-lomerase activity in vitro (52). The tertiarystructure of the minimal hTR pseudoknotalso provided a structural explanation forthe effect of disease mutations in this re-gion of hTR (52, 54). Mutational studiesand modeling of yeast telomerase pseu-doknot domains (53, 80) have providedadditional evidence for an essential rolefor a conserved triple helix in the pseu-doknot domain. The putative pseudoknotsin ciliates are much smaller than thosepseudoknots in yeasts and vertebrates.Although no structures of ciliate pseu-doknots have yet been reported, sequenceanalysis and modeling of 28 ciliate TRshave predicted one (and in one case, two)potential base triples in the ciliate pseu-doknots (81).In the minimal hTR pseudoknot

(PKDU), the bulge U177 was removed tostabilize the pseudoknot formation. How-ever, deletion of this residue in hTRresulted in a 2- to 10-fold decrease intelomerase activity (52, 78), and compu-tational modeling of the P2b-P3 pseu-doknot suggested that deletion of U177alters tertiary interactions within thepseudoknot (82). In addition, substitutionof the 2′-OH of residue A176, which isadjacent to residue U177, with 2′-OMe or

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Fig. 1. Secondary structure and known proteincomponents of the human telomerase holoen-zyme. The human telomerase RNA (hTR) containsthree major structural and functional domains,the core domain, the CR4/CR5 domain, and the H/ACA scaRNA domain (46, 59, 62). The hTR core andCR4/CR5 domains independently bind the hTERT(blue ellipse) (50, 64). The hTR scaRNA domainbinds two sets of the four H/ACA RNP proteins:dyskerin (green), Gar1 (cyan), Nop10 (magenta),and Nhp2 (orange) (67). The protein TCAB1/WDR79 (purple) binds both the dyskerin and theCAB box located at the CR7 region within the H/ACA scaRNA domain (72, 73).

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2′-H results in about a twofold decrease inactivity, leading to the proposal that theA176 2′-OH may contribute directly totelomerase catalysis (53). To providestructural insight into the WT P2b-P3pseudoknot (PKWT) and the functionalrole of U177, the solution structure ofPKWT was determined, the originalstructure of PKDU was further refinedwith an extensive set of NMR residualdipolar couplings (RDCs) (Fig. 2B), andsystematic structural and dynamical com-parisons were made between PKWT andPKDU (54). These structural character-izations revealed that PKWT folds into thesame H-type pseudoknot conformation asPKDU with almost identical tertiary in-teractions (Figs. 2B and 3 A and B). Inboth PKWT and PKDU, the P2a and P2bhelices are stacked on either side of

a junction that is a Hoogsteen base pairformed between the first nucleotide (U99)in the J2b/3 loop and the last nucleotide(A173) in the J2a/3 loop, and both pseu-doknots are stabilized by the same basetriples flanking this junction base pair(Figs. 2B and 3B). The A-rich J2a/3 loopforms two minor groove base triples withthe P2b helix, and the U-rich J2b/3 loopforms three major groove U-A·U Hoogs-teen base triples with P3 helix (Fig. 3C).However, PKDU is also stabilized by anadditional C112·G178-U103 triple that isdisrupted in PKWT because of the pres-ence of U177, which creates a large rolland tilt between the flanking base pairs(Fig. 3 A and D). In PKWT, U177 is flip-ped out of the P3 helix into the minorgroove and is located on the opposite sideof the helix from the major groove triple

helical interactions. Based on the struc-ture, the base of U177 sits right over the2′-OH of residue A176 and would steri-cally block its accessibility during telo-merase catalysis (Fig. 3D). Dynamiccharacterization by NMR spin relaxationmeasurements revealed that U177 is in-trinsically highly flexible, which suggeststhat, rather than blocking A176, U177 mayserve as a hinge to provide additionalbackbone flexibility for residue A176 tofacilitate the catalysis (54).The structure determination of PKWT

also revealed why it is thermodynamicallyless stable than PKDU (52). Comparisonof NMR imino proton spectra of PKWTand PKDU as a function of temperaturerevealed an unanticipated difference inthe late folding/early unfolding pathway.In PKDU, as expected, when temperatureincreases, the Hoogsteen base pairs be-tween the J2b/3 loop and P3 helix begin tomelt before the Watson–Crick base pairsin P3 helix. However, in PKWT, thepresence of bulge U177 results in partialunstacking of the three Watson–Crick U-A base pairs from the rest of the helixbelow the bulge U177 (Fig. 3A). As aconsequence, the three U-A base pairsabove and the two base pairs below thebulge U are thermodynamically less stablethan the tertiary (Hoogsteen base pair)interactions, and they begin melting beforethe Hoogsteen U-A base pairs. Thus, thepresence of U177 in PKWT results in analtered late folding/early unfolding path-way in the pseudoknot, which may be rel-evant to RNA assembly. In addition, forboth pseudoknots, the melting studies re-vealed that the junction J2b/3 loop–J2a/3loop Hoogsteen U-A base pair is

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Fig. 2. Structures of subdomains of the hTR core domain. (A) Sequence and secondary structure of the hTR core domain, which can be divided into foursubdomains: P2a-J2a.1-P2a.1 (blue-gray-gold), P2a-J2a/b-P2b (gold-green-red), P2b-P3 pseudoknot (red-pink), and P1 (dark green) (46). (B) NMR solutionstructures of the P2b-P3 pseudoknot, where PKDU (PDB ID 2K96) and PKWT (PDB ID 2K96) are structures for ΔU177 mutant and WT constructs, respectively(54). (C) NMR solution structure of P2a-J2a/b-P2b (PDB ID 2L3E) (55). (D) Structural model of the P2a-J2a.1-P2a.1 determined by the RDC-MC-Sym approach (55).All 3D structures are color-coded like the secondary structure in A, and nonnative residues are colored light gray.

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Fig. 3. Tertiary interactions in the pseudoknot. (A) The structure of the triple helical region of PKWT(54). (B) Schematic representations of the tertiary interactions in PKWT. In PKDU, U103 forms a Hoogs-teen base pair with G178. (C) U·A-U Hoogsteen base triple. (D) Detailed view of the base pairs sur-rounding U177. The base of U177 stacks over the 2′OH of A176. The nucleotides are in CPK colors, exceptthat U177 is colored magenta.

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remarkable stable. Thus, this base pairplays a critical role in overall folding of thepseudoknot (54).

Structure, Dynamics, and Function of the P2a-J2a/b-P2b.Adjacent to the highly conservedP2b-P3 pseudoknot is a 5-nt bulge loop,J2a/b, which serves as a bridge between thepseudoknot and the P2a helix. This py-rimidine-rich J2a/b bulge loop is not highlyconserved in sequence, except for a rela-tively conserved G at the 5′ end (61% invertebrate and 83% in mammalian TRs)(39). Initial studies showed that swappingthe bulge sequence 5′→3′ or replacing thehTR sequence with the mouse TR se-quence had little effect on human telo-merase catalytic activity (63, 83). Theseresults, along with the lack of sequenceconservation, argued against an importantrole of the J2a/b bulge loop in telomeraseactivity. However, the location of theJ2a/b bulge loop is conserved in all verte-brate TRs, and its length is usually 5 nt inmammalian TRs (46, 47). The solutionstructure of the J2a/b bulge loop togetherwith flanking P2a and P2b helices showedthat J2a/b introduces a large bend (89 ±3°) between P2a and P2b across the majorgroove (Fig. 2C) (55). The 5′-end residueof the bulge loop, G84, stacks above P2a,and the 3′-end residue of the bulge loop,C88, stacks below P2b. A change in thebackbone direction occurs at the centerbulge residue, U86, which leads to anoverall S-shape conformation of J2a/b.Remarkably, the S-shape structure of theJ2a/b results in almost no twist betweenP2a and P2b (−10 ± 10°) (Fig. 2C). Asearch for other 5-nt bulge structures, us-ing the FRABASE program (84), un-covered only one other 5-nt bulge amongall RNA structures solved to date from thehepatitis C virus (HCV) internal ribosomeentry site (IRES) domain II (85). Evenmore surprisingly, this HCV IRES domainII bulge adopts the same S-shaped struc-ture, despite the fact that it has a differentsequence from the hTR J2a/b. A some-what broader search that also allowed fornoncanonical closing base pairs andswapping the strand with the 5-nt bulgeuncovered two additional sequences, andthese sequences have similar S-shaped in-ternal loops (86, 87). Thus, the J2a/b bulgerepresents a rare structural motif.Systematic dynamic characterizations by

NMR 15N spin relaxation (88) and NMRRDCs (89, 90) also revealed another un-usual feature of the J2a/b bulge loop. Itwas initially expected that the J2a/b bulgeloop would be highly flexible, but althoughJ2a/b exhibits some flexibility, its motion isremarkably limited on the nanosecond tomillisecond time scale compared with the3-nt bulge loop in HIV-1 transactivationresponse element (TAR) RNA. Based ona cone motional model, P2a and P2b move

relative to each other with a motionalamplitude of ∼39°. In contrast, HIV-1TAR RNA has a cone-model motionalamplitude of ∼55° (91).The rare structure and unexpected dy-

namics of J2a/b suggested that it mighthave an important functional role in telo-merase RNA topology and function. Totest this hypothesis, systematic mutationswere carried out to investigate the effectsof the length, strand location, and sequenceof J2a/b on telomerase function (55). Theresults showed not only that the di-rectional bending defined by J2a/b is re-quired for overall telomerase activity butalso that the intrinsic flexibility across J2a/b is important for processive catalysis bytelomerase. The results also suggest theconsensus sequence G83Y78Y87Y96Y87 forthe 5-nt bulge in mammalian TRs mayhave evolved for both nucleotide additionand template translocation during telo-merase catalysis. Finally, the large bend atthe J2a/b bulge would be expected to playa significant role in determining the over-all topology of the core domain, whichdiscussed more below.

Structure and Dynamics of the Mammalian-Specific P2a.1-J2a.1-P2a. Located on theother side of hTR P2/P3 pseudoknot is theP2a.1-J2a.1-P2a domain, where P2a.1 isa mammalian-specific extension to the P2ahelix through an asymmetric internal loopJ2a.1. Biochemical studies have shown thatsome nucleotide substitutions in this regionand the C72G mutation associated withaplastic anemia result in a decrease intelomerase activity (62, 63, 83). In NMRspectra of a P2a.1-J2a.1-P2a construct,most of the nonexchangeable protonsfrom the J2a.1 loop and surrounding nu-cleotides were exchange-broadened and inmany cases, not visible. This finding notonly indicated the presence of significantconformational exchange in J2a.1, appar-ently because of the bases in the internalloop adopting more than one conforma-tion involving alternative base pairs, butalso hindered high-resolution structuredetermination of this region of hTR bysolution NMR (55). To overcome thesedifficulties, the RDC-MC-Sym approachwas developed for structure determinationof nucleic acids (55). This approach usesa combination of computational modelingby program MC-Sym (92) and experi-mentally derived restraints by NMRRDCs. The structural analysis revealedthat P2a.1 is essentially a linear extensionof the P2a helix (Fig. 2D). The interhelicalbend between P2a.1 and P2a is only 6 ± 3°,and the twist between the two helices is135 ± 14°, which agrees well with theamount of twist for an ∼4-bp irregularhelix formed by the asymmetric loop J2a.1.Although J2a.1 undergoes significantconformational exchange, the interhelical

motion between P2a.1 and P2a is re-markably restricted (∼26° cone motions)based on dynamic characterization byNMR RDCs. The stability betweenhelices is consistent with the observationsthat disruption of base pairs flankingJ2a.1 decreases telomerase activity andthat nonmammalian vertebrate TRshave a single long P2a helix without aJ2a.1 (46, 47).

High-Resolution Model of the hTR Core Do-main. The structures and dynamic anal-ysis of subdomains of the hTR core do-main, in particular, the conservedpseudoknot, have provided great insightsinto their functional roles in telomerasecatalysis. However, they do not provide anoverview of how the P2/P3 pseudoknotfolds and how its architecture imposesconformational constraints that positionthe template into the active site of telo-merase. A low-resolution (6.5–8.0 Å)FRET model of the hTR core domain hasbeen reported (PDB ID 2INA), wheredistances derived from FRET betweenfluorescently labeled peptide nucleic acidswere used in structural modeling (93). Inthis approach, although these distanceswere obtained in the full-length hTR, theyprovide only indirect constraints on thepositions of the helical regions of the hTRcore domain, because the peptide nucleicacids were hybridized onto three single-stranded regions, the 5′ end of hTR (nu-cleotides 1–13), the template region (nu-cleotides 44–56), and the J2a/3 region(nucleotides 146–158). One caveat is thatthe inherent flexibility of the single-strandregions, in particular, at the 5′ end of thehTR, was not considered in the modeling.Recently, by applying a computationalmodeling approach that incorporates theAssisted Model Building with Energy Re-finement (AMBER) force field and short-range NOE restraints from individualstructures, a high-resolution structuremodel of the P2/P3 pseudoknot was de-termined that contains all nucleotides ex-cept for the nonconserved single-strandregion of J2a/3 (Fig. 4A) (55). There aresignificant differences between the FRET-and NMR-based structure models in boththe local structures and relative helicalorientations, but both models do exhibitan open architecture of the hTR core do-main and a major interhelical bend oc-curring across the J2a/b bulge (55, 93).The NMR-based structure model

revealed that the full-length P2/P3 pseu-doknot of the hTR core domain folds intoan overall V-shape conformation that isdefined by the ∼90° bend across the J2a/b bulge. The P2b-P3 pseudoknot andP2a.1-J2a.1-P2a domain are located oneither side of the J2a/b bulge, and they areeach ∼50Å long. Thus, the bend acrossJ2a/b creates an ∼70 Å end to end

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distance between P2b-P3 and P2a.1, whichagrees well with the length of the in-tervening 24-nt single strand containingthe template without significant stretching.Because of the negligible twist betweenthe P2a and P2b induced by the J2a/b bulge loop, the structural model alsorevealed that the conserved bulge residueU177 (52, 78) and the catalytically im-portant 2′OH of residue A176 (53) areboth positioned on the inner surface of thecore domain and face to the 5′ end of thetemplate, where the active site should belocated. The structural model is also con-sistent with the close proximity of theend of the pseudoknot to the templateneeded to obtain maximal activity in anengineered cis-telomerase (94). In addi-tion, the dynamic characterization showedthat interdomain motion between P2a andP2b has an amplitude of ∼39° assuminga cone motional model, which can betranslated into a displacement of ∼28 Åbetween the two ends in the full-length P2/P3 pseudoknot. This distance agrees wellwith the ∼17 Å that the template musttranslocate through the active site duringthe synthesis of each TTAGGG telomererepeat. Although the binding sites ofTERT on the core domain have not beenidentified, the overall shape of the P2/P3pseudoknot fits nicely into the crystalstructure of the TERT from T. castaneum(36, 37). The pseudoknot can be modeledonto the TERT either in parallel or per-pendicular to where the template/primerfits in the donut hole of the TERT, suchthat flexing of J2a/b would allow the tem-plate to be pulled through the active siteduring telomere synthesis (Fig. 4B). Thisfinding provides a testable model for howthe core domain interacts with TERT.

STE of hTRThe STE is the other catalytically essentialTR element conserved across ciliates,vertebrates, and yeasts (9, 50, 95, 96). In

hTR, the CR4/CR5 domain is the STE,and it interacts directly with hTERT in-dependently from the core domain. ThehTR CR4/CR5 domain contains a three-way junction (Fig. 5A), which has alsobeen proposed to form in yeast telomeraseRNAs (97). Like the core domain, al-though the CR4/CR5 secondary structureis conserved, most conserved residues arelocalized in one particular region, the P6.1hairpin. Additional highly conserved resi-dues are located at the large internal loopthat forms a three-way junction betweenP6, P6.1, and P5. Although various Wat-son–Crick base pairs can be drawn be-tween nucleotides in the three-wayjunction, substitutions and compensatorymutations indicate that it is the sequencerather than the base pairs that are impor-tant for telomerase activity (98). In addi-tion, a minimal CR4/CR5 domainconstruct, which includes P6.1, all of P6a-P6b except the terminal hairpin, andflanking nucleotides but does not includethe P5 helix, is sufficient to reconstitutetelomerase activity in trans with the coredomain and TERT (Fig. 5A) (76). Thus, itis not clear whether this region formsa three-way junction in the context of itsassociation with TERT. To date, nostructural information on the CR4/CR5three-way junction has been reported, butthe structures of two helical regions of thehTR STE have been solved (74–76). Thesestructures are the essential P6.1 hairpinand the central portion of P6 surroundingthe J6 internal loop.

Structures of P6.1 and Pseudouridylated P6.1The P6.1 is a short hairpin off of the three-way junction that has a helix of fourWatson–Crick base pairs capped by a 5-ntloop. The 13-nt sequence of P6.1 is highlyconserved; in addition to U307 and G309in the loop, all 8 nt forming Watson–Crickbase pairs are 100% conserved in verte-brates. The formation of base pairs in P6.1

has been shown to be essential for TERTbinding and telomerase activity (50, 98).Both the length of the P6.1 helix and thetwo conserved loop residues are critical fortelomerase activity but not for binding ofTR to TERT (64, 98). The structure ofP6.1 showed that the first (U306) and thelast (G310) residues of the 5-nt loop forma U-G wobble pair on top of the A-formhelix (Fig. 5B) (75). The remaining threeresidues from the loop, U307, G309, andG310, are exposed to solvent, with U307and G309 located on the minor grooveside of the loop and G308 located on themajor groove with partial stacking onU306. The small loop formed by thesethree residues has a well-defined confor-mation, and this architecture of the loophas been proposed to allow the essentialloop nucleotides to interact with TERT orTR (75). Intriguingly, gel shift and cross-linking experiments have shown that iso-lated hTR fragments of P6.1 and thetemplate can directly interact with con-tacts between the P6.1 loop residues andthe two ends of the template region (99),although the biological relevance of thisinteraction in the context of hTERT re-mains to be established.Most noncoding RNAs contain modified

nucleotides, and hTR seems to be no ex-ception. Six potential pseudouridine (Ψ)modification sites have been identifiedwithin the hTR, two of which are locatedin the P6.1 loop (76). Pseudouridine is themost abundant posttranscriptional modi-fied RNA nucleotide, and it is found in allspecies (100). The locations of pseudour-idines are usually highly conserved, andthey play important roles in biologicalfunctions. To investigate the potentialfunctional roles of these Ψ-modificationsin the essential P6.1 loop, the solutionstructure of a pseudouridine-modifiedP6.1 (Ψ-P6.1) was determined (Fig. 5B)(76). Furthermore, both the thermody-namic stability of the Ψ-P6.1 vs.

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Fig. 4. Models of the hTR core domain and interaction with TERT. (A) NMR-based model of the hTR core domain including a DNA primer bound to thetemplate (55). (B) The hTR P2/P3 pseudoknot positioned onto the T. castaneum TERT in two possible orientations, where the hTR P2/P3 pseudoknot lies eitherparallel (Left) or perpendicular (Right) to the T. castaneum TERT-telomeric RNA/DNA complex (PDB ID 3KYL) (37). The color scheme for the hTR P2/P3 pseu-doknot domain is the same as in Fig. 2. Domains of the T. castaneum TERT are colored and labeled as shown, and the RNA template and telomeric DNA arecolored in purple and cyan, respectively. A comparison of the domain structures of the hTERT and T. castaneum TERT is also shown.

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unmodified P6.1 and the effect of pseu-douridylation on telomerase activity in vi-tro were characterized. The loop structureof the Ψ-P6.1 is significantly different fromthe loop structure of P6.1. The stem clos-ing Ψ306·G310 base pair has a single hy-drogen bond between the imino proton ofG310 and the O4 of Ψ306, which is dif-ferent from the canonical U·G wobble pairfound in the unmodified P6.1. The basesof Ψ306 and Ψ307 are located in the majorgroove of the loop and are stacked onG305 and Ψ306, respectively. These twopseudouridines are spatially positioned toform water-mediated hydrogen bonds be-tween the imino protons and their 5′phosphate oxygens. Only one base, thenonconserved G308, is flipped out of theloop, and it is on the minor groove side.The phosphate backbone turns betweenG308 and G309, and G309 in the synconformation stacks on the sugar of G310.These structural features revealed that theΨ-modifications at the loop increase bothbase-stacking and hydrogen-bonding in-teractions in the Ψ-P6.1 relative to theunmodified P6.1, resulting in higher ther-modynamic stability that is characterizedby UV melting studies. Interestingly, thepseudouridine modification at 306 and 307in the P6.1 loop decreased the telomeraseactivity in vitro by approximately threefoldbut slightly increased the telomere addi-tion processivity (∼20%), indicating that

pseudouridylation may have a subtle butsignificant effect on telomerase activity.

Structure of the P6 Hairpin. The P6 hairpin islocated adjacent to the 5′ end of P6.1. Ithas two helical regions, P6a and P6b, thatflank a small asymmetric internal loop J6,and it is capped by a UCCG hairpin (Fig.5A). Deletion of the hairpin loop has noeffect on telomerase activity, but addi-tional deletions that include the J6 in-ternal loop have been shown to inhibitinteractions between CR4/CR5 and TERTand abolish telomerase activity (50).The solution structure of an RNA de-

rived from P6, including the J6 internalloop, has been determined (Fig. 5C) (74).P6a was predicted on the basis of phylo-genetic analysis to have a C262-A295 basepair next to bulge U261, but in the struc-ture, a U261-A295 base pair forms andC262 is bulged out. The J6 internal loopis well-defined, with a potential tripleformed between C267 through water-mediated hydrogen bonds to the G268-C288 base pair. Interestingly, similar to theP2a.1-J2a.1-P2a, the interhelical motionbetween P6a and P6b across this asym-metric bulge is very limited with amplitude<10°, which was revealed by RDC analysis.These structural and dynamical propertiesof J6 result in a unique conformation ofthe P6 region. In addition to a 20° inter-helical angle and ∼3 Å deflection betweenP6a and P6b, the J6 internal loop forms anunusual solvent-accessible opening, lead-ing Leeper et al. (74) to speculate that thisinternal loop may be important for TERTor TR interactions. However, a variety ofbase substitutions in this internal loop donot abrogate activity (50), and therefore, itis not clear at the current stage that thisstructure is unique.

H/ACA scaRNA Domain of hTRIn vivo biogenesis and regulation of telo-merase holoenzyme require additionaltelomerase RNAmotifs. In vertebrates, the3′-half of TR comprises an H/ACA scaR-NA domain, which includes a telomerase-specific CR7 domain that forms a 3′-ter-minal hairpin. All H/ACA small nucleolarRNAs (snoRNAs) and scaRNAs formRNPs with evolutionary conserved H/ACA RNP proteins and generally functionto direct site-specific pseudouridylation ofribosomal RNAs and small nuclear RNAs,respectively (101–104), but in the case oftelomerase, the domain seems to havebeen co-opted to help localize telomeraseto Cajal bodies (56, 58, 59). The H/ACAmotif has a hairpin-hinge-hairpin-tail sec-ondary structure, where the conserved Hbox is located at the hinge region and theACA box is located at the 3′-end tail (66).In addition to the H/ACA motif, scaRNAsshare another common motif, known asthe CAB box, which has a consensus

ugAG sequence and serves as a CB local-ization signal (105). Remarkably, the CR7domain of vertebrate TRs, as discussedbelow, contains not only a CB localizationsignal but also another signal for the ac-cumulation and processing of TRs (60).Although the H/ACA and CR7 domainsare not required for telomerase activityin vitro, they form a scaRNA domainthat is essential for in vivo accumulation,3′-end processing, and localization ofvertebrate TRs.Although no high-resolution structures

of the H/ACA domain from any verte-brate TRs with or without H/ACA RNPproteins have been determined to date,a combined structural and biochemicalstudy, including localization studies usingFISH, on the hTR has delineated the

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Fig. 5. Structures of subdomains of the hTR CR4/CR5 domain. (A) Sequence and secondary struc-ture of the hTR CR4/CR5 domain (46). The minimalCR4/CR5 required for reconstitution in vitro ofactive telomerase is highlighted by a dashed box(76). (B) NMR solution structures of P6.1 (PDB ID1OQ0) (75) and Ψ4P6.1 (PDB ID 2KYE) (76). Thetwo U and three G letters in the P6.1 loop arecolored in green and yellow, respectively. The twopseudouridines (Ψs) in the P6.1 loop are colored inred. (C) NMR solution structure and secondarystructure of a P6 hairpin construct (PDB ID 1Z31)(74). The internal loop residues (C266-C267 andA289-U291) are colored in green, and the bulgeresidue C262 is colored in purple.

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Fig. 6. Structure and function of the hTR CR7domain (60). (A) Sequence and secondary struc-ture of the hTR CR7 hairpin and flanking basepairs. Boxed residues are the CAB box sequence.Residues with >95% and >85–95% conservationamong all vertebrate species are shown in capitalletters and bold fonts, respectively. Nonnativeresidues used in structure determination are thebase pairs below the dashed line. (B) NMR solutionstructure of the CR7 hairpin (PDB ID 2QH2). Resi-dues are colored by type: orange, A; green, U;blue, G; red, C. (C and D) Residues comprising thehTR-specific processing signal are highlighted inmagenta in the secondary structure (C) and col-ored as in B in the surface representation of theCR7 structure (D). (E and F) Residues comprisingthe CB localization signal are highlighted in darkblue (CAB box) and light blue in the secondarystructure (E) and colored as in B in the surfacerepresentation of the CR7 structure (F).

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sequence and structural elements of thetwo independent signals, located in CR7,for 3′-end processing and accumulationand localization of hTR to Cajal bodies(Fig. 6) (60). The CR7 hairpin loop hasfour key structural features. First, withinthe 8-nt loop, there is 1 bp observed be-tween the first and the second to last nu-cleotides of the loop, a U411·G417 wobblepair. Second, because of the U·G pair inthe loop and stable Watson–Crick basepairs in the helix, the last nucleotide of theloop, U418, adopts an unpaired confor-mation. Third, the first 3 loop nt that arealso the first 3 nt of the CAB box arestacked over each other, forming A-formbackbone geometry. Finally, although thelast CAB box residue (G414) does notstack over other residues and is quiteflexible, a change in the backbone di-rection occurs between G414 and C415,positioning all CAB box residues on the 5′side of the loop. To determine which ofthese structural features is important forthe 3′-end processing and localizationsignals, parallel structural determinationswere performed on the terminal loopconstruct derived from the human U64 H/ACA snoRNA and human U85 C/D-H/ACA scaRNA. It had been previouslyshown the U64 snoRNA replacement ofthe hTR CR7 in the full-length hTR al-lows hTR accumulation and processing,but the RNA localizes to nucleoli insteadof Cajal bodies. In contrast, replacementof the CR7 hairpin with the 3′ hairpin ofU85 scaRNA abrogated processing, andthe hTR remained at the site of tran-scription. Comparison of the terminal loopstructures of the hTR CR7 and the 5′hairpin of human U64 H/ACA snoRNA

revealed that the first two structural fea-tures described above are shared in com-mon between these two RNAs, andadditional mutational analysis confirmedthat the processing signal for 3′-end mat-uration of hTR comprises a U-G base pairbetween the first nucleotide of the loopand the penultimate nucleotide of theloop, an unpaired U418 at the last positionof the loop, and some sequence specificityfor the top 2 bp in the helix (Fig. 6 C andD). Comparison of the structures of thehTR CR7 hairpin loop and the humanU85 C/D-H/ACA scaRNA, combined withcharacterization of the processing, locali-zation, and accumulation of various mu-tant and Wt RNAs, revealed that the CBlocalization signal comprises the ugAGCAB box nucleotides as well as a base-paired helix, with some dependence of thesequence identity of the top 2 bp (Fig. 6 Eand F). Recently, the protein responsiblefor binding to the CB localization signaland driving telomerase and other H/ACAscaRNAs to Cajal bodies has been identi-fied as TCAB1/WDR79 (72, 73). It is in-teresting to note that, although they areboth in the same hairpin, the CB locali-zation signal and the 3′-end processingsignal could potentially independently andsimultaneously bind TCAB1/WDR79 andthe unidentified protein that is requiredfor TR accumulation and 3′-end process-ing. Furthermore, the 3′ processing signalis the same site where the H/ACA RNPprotein Nhp2 would be expected to bind(60, 106).

SummaryDuring the past decade, combined use ofsecondary structure predictions, muta-

tional analysis, structure determination ofsubdomains, and structure-based model-ing of hTR in conjunction with bio-chemical characterization have revealedessential elements of hTR structure andfunction beyond the role in templating thetelomere DNA. Important insights in-clude the identification and character-ization of an essential triple helix in theconserved pseudoknot in the core do-main, a structurally conserved bulgeloop in the core domain that directs theoverall topology, and two independentsignals, for Cajal body localization andhTR 3′-end processing, in the 3′-terminalhairpin of hTR. Recent structural studiesof the core domain have provided atestable model structure of the core do-main that is consistent with all currentbiochemical data. However, much re-mains to be learned, particularly abouthow and where hTERT interacts withthe core domain and how the core do-main and the STE interact with hTERTand potentially each other to facilitatecatalysis. The structure of the H/ACAscaRNA domain and how two sets ofH/ACA RNP proteins bind two con-secutive hairpin-bulge-hairpin sequencesremain to be determined. Combineduse of NMR, X-ray crystallography,small-angle X-ray scattering, single mol-ecule techniques, and EM will likely re-veal these structures within the nextfew years.

ACKNOWLEDGMENTS. Q.Z. is a Baltimore FamilyFellow of the Life Sciences Research Foundation.This work was supported by grants from the Na-tional Institutes of Health and the National ScienceFoundation (to J.F.).

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20332 | www.pnas.org/cgi/doi/10.1073/pnas.1100279108 Zhang et al.

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