Structural Basis for Telomerase RNA Recognition and RNP Assembly by the Holoenzyme La Family Protein p65

Molecular Cell

Article Structural Basis for Telomerase RNA Recognition and RNP Assembly by the Holoenzyme La Family Protein p65 Mahavir Singh,1 Zhonghua Wang,1 Bon-Kyung Koo,1 Anooj Patel,1 Duilio Cascio,1 Kathleen Collins,2 and Juli Feigon1,* 1Department of Chemistry and Biochemistry, and the Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095-1569, USA 2Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3200, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2012.05.018

SUMMARY

Telomerase is a ribonucleoprotein complex essential for maintenance of telomere DNA at linear chromosome ends. The catalytic core of Tetrahymena telomerase comprises a ternary complex of telomerase RNA (TER), telomerase reverse transcriptase (TERT), and the essential La family protein p65. NMR and crystal structures of p65 C-terminal domain and its complex with stem IV of TER reveal that RNA recognition is achieved by a combination of singleand double-stranded RNA binding, which induces a 105
bend in TER. The domain is a cryptic, atypical RNA recognition motif with a disordered C-terminal extension that forms an a helix in the complex necessary for hierarchical assembly of TERT with p65TER. This work provides the first structural insight into biogenesis and assembly of TER with a telomerase-specific protein. Additionally, our studies define a structurally homologous domain (xRRM) in genuine La and LARP7 proteins and suggest a general mode of RNA binding for biogenesis of their diverse RNA targets.

INTRODUCTION Telomerase is an RNA-protein particle (RNP) that extends the G-rich strands of the telomere repeat DNA at the ends of linear chromosomes (Blackburn and Collins, 2011; Greider and Blackburn, 1985). Most somatic cells have low levels of telomerase activity, resulting in telomere attrition due to incomplete replication of telomere ends, which leads to cellular senescence or apoptosis (Bertuch and Lundblad, 2006; Shay and Wright, 2011; Wong and Collins, 2003). In contrast, most cancer cells have high levels of telomerase activity, an important element of cancer etiology (Shay and Wright, 2011). Telomerase insufficiency due to mutations in telomerase components that affect biogenesis or catalysis has been linked to dyskeratosis congenita, aplastic anemia, pulmonary fibrosis, and other diseases (Artandi and DePinho, 2010; Chen and Greider, 2004; Walne 16 Molecular Cell 47, 16–26, July 13, 2012 ª2012 Elsevier Inc.

and Dokal, 2009). Telomerase is composed of telomerase RNA (TER), telomerase reverse transcriptase (TERT), and several additional proteins required for assembly and activity in vivo (Blackburn and Collins, 2011; Podlevsky and Chen, 2012). TER provides the template for the telomere synthesis and plays essential roles in assembly, localization, accumulation, and catalytic activity (Cunningham and Collins, 2005; Theimer and Feigon, 2006; Zhang et al., 2011). TERT has the active site along with RNA and DNA binding activities (Autexier and Lue, 2006; Blackburn and Collins, 2011; Lingner et al., 1997; Mason et al., 2011). The structural roles and mechanism of the telomerasespecific accessory proteins in biogenesis, assembly, and catalysis remain largely unknown. Tetrahymena p65 is a telomerase holoenzyme protein essential for TER accumulation in vivo (Witkin and Collins, 2004). In the Tetrahymena holoenzyme, TER, TERT, and p65 comprise a stable catalytic core complex, and p75, p45, and p19 comprise a telomere adaptor subcomplex (TASC) (Min and Collins, 2009, 2010; Witkin and Collins, 2004). Formation of the catalytic core complex is independent of TASC assembly, which is required for telomere maintenance (Min and Collins, 2009). Biochemical and FRET studies have shown that hierarchical assembly of the catalytic core starts with the binding of p65 to TER, which results in a conformational change in TER that facilitates the binding of TERT to the p65-TER complex (O’Connor and Collins, 2006; Prathapam et al., 2005; Stone et al., 2007). p65 and its ortholog from the ciliate Euplotes aediculatus, p43 (Aigner et al., 2003), have been classified as La-related proteins group 7 (LARP7) (Bousquet-Antonelli and Deragon, 2009). Genuine La and LARP proteins are RNA chaperones for diverse RNA targets, including mRNA, tRNAs, snRNAs, snoRNAs, and 7SK RNA (Wolin and Cedervall, 2002; Bayfield et al., 2010; Bousquet-Antonelli and Deragon, 2009). Four domains of p65, N-terminal, La motif (LAM), RNA recognition motif (RRM), and C-terminal (Figure 1A), have been proposed to interact with specific sites in stem I and stem-loop IV of TER (Figure 1B), and binding of the unique C-terminal domain of p65 in particular requires the region of stem IV around the central dinucleotide GA bulge (Akiyama et al., 2012; O’Connor and Collins, 2006; Prathapam et al., 2005). p65 also stimulates conformational changes in other parts of TER, and it stabilizes TER and TERT in catalytically active conformations (Berman et al., 2010). Interaction of the p65 C-terminal domain with TER is necessary and sufficient for the

Molecular Cell Structure of p65 xRRM2 Complex with Telomerase RNA

Figure 1. Structure of p65 C-Terminal Domain (A) Domain structure of p65 and p65 C-terminal domain constructs. (B) Sequence and secondary structure of Tetrahymena TER and stem IV constructs. TBE indicates the template boundary element. (C–E) Solution NMR structure of p65-C2DL1: (C) ensemble of the 20 lowest energy NMR structures, (D) lowest energy structure; side view of b sheet. Atypical features are highlighted: a3 (red), b40 (lime green), long b2-b3 loop (gray), nonaromatic residues at conserved positions on b3 RNP1 (cyan) and b1 RNP2 (violet), and location of the start of the C-terminal tail (red +). (E) Cartoon rendering of lowest energy structure. The position of the b2-b3 loop is indicated by gray dots. (F) Crystal structure of p65-C1DL2 in cartoon rendering. In (C)–(F), the b sheet is orange, a3 is red, the b2-b3 loop is gray, and the rest of the protein is light orange.

hierarchical assembly of the TERT-TER-p65 catalytic core (Akiyama et al., 2012; O’Connor and Collins, 2006; Prathapam et al., 2005). Here we report the structure of a complex of a telomerase-specific protein with telomerase RNA. Analysis of the structures of p65 C-terminal domain free and in complex with stem IV of TER and biochemical data reveal how p65 functions in telomerase biogenesis and assembly. Our studies define a new class of RRM with a C-terminal extension that forms a helix in complex with RNA (xRRM) and suggest that its mode of RNA binding is common to genuine La and LARP7 proteins for biogenesis of their diverse targets. RESULTS AND DISCUSSION p65 C-Terminal Domain Is a Cryptic Atypical RRM Although the p65 C-terminal domain binds RNA, global BLAST searches for homologous sequences and putative domains using the p65 C-terminal domain did not identify a known protein fold. To define the domain boundaries, we made a series of C-terminal domain constructs (Figure 1A and see Figure S1 online). p65-C(369–542) (hereafter, p65-C1) gave a protein with a reasonably good 1H-15N HSQC spectrum indicative of a folded protein (Figure S1C). Analysis of this spectrum indicated that 22 residues at the C terminus were disordered, so these were deleted to make p65-C2 (Figure 1A and Figure S1D). Lastly,

internal residues 421–442 were deleted to make p65-C1DL1 (residues 369–542D421–442) and p65-C2DL1 (residues 369– 519D421–442), respectively (Figure 1A and Figures S1F and S1G), since residues 413–459 exhibited few NOEs, and chemical shift indexing and heteronuclear NOE analysis (Figure S1E and see the Experimental Procedures) indicated that this region of the protein is random coil. Comparison of the 1H-15N HSQC spectra of p65-C1DL1 and p65-C2DL1 with p65-C1 and p65C2 spectra, respectively (Figures S1C, S1D, S1F, and S1G), showed that the internal loop deletion did not change the folding of the domain. As shown below, deletion of these internal residues also did not significantly affect RNA binding. A solution NMR structure of p65-C2DL1 was solved to an rmsd of 0.26 A˚ for the 20 lowest energy structures (Figure 1C and Table S1). The structure reveals that the overall fold of p65 C-terminal domain is an RRM, but with several atypical features (Figure 1D). The p65-C2DL1 RRM has a b1-a1-b2-b3-a2-b40 b4-a3 topology, with a five-stranded antiparallel b sheet (Figures 1C–1E). Atypical features of p65 C-terminal RRM are the presence of a short b40 strand; the absence of the consensus RNP1 and RNP2 sequences on the b3 and b1 strands, respectively, with three aromatic residues that generally recognize two nucleotides; and a noncanonical third helix, a3 (residues 504–517) that lies directly across the middle-upper surface of the ‘‘RNA binding’’ face of the b sheet (Figures 1C and 1D). Molecular Cell 47, 16–26, July 13, 2012 ª2012 Elsevier Inc. 17

Molecular Cell Structure of p65 xRRM2 Complex with Telomerase RNA

Figure 2. Isothermal Calorimetry of p65 C-Terminal Domain with TER Stem IV (A–F) Isothermal calorimetry data and analysis for titration of S4 RNA into (A) p65-C1, (B) p65-C1DL1, (C) p65-C1DL2, (D) p65-C2, (E) p65-C1DL2 Y407A, and (F) p65-C1DL2 R465A. ITC data for other constructs are shown in Figure S5.

ture (Figure 1E), except that the b2 and b3 strands are each one residue shorter at the b2-b3 end and there is a small difference in the position of the b sheet (Figure S2C). Significantly, no electron density is observed for the C-terminal tail of the RRM (after residue 517), which NMR analysis indicates is unstructured in solution.

C-terminal a helices have been observed in some RRMs, but with varying orientations and proposed functional roles (Alfano et al., 2004; Allain et al., 1996; Clery et al., 2008; Jacks et al., 2003; Martin-Tumasz et al., 2011; Oubridge et al., 1994; Varani et al., 2000). An unusual feature of p65 C-terminal RRM is the presence of the 47 residue (413–459) loop between b2 and b3 (b2-b3 loop), which in p65-C2DL1 was truncated to 21 residues (Figures 1A and 1D and Figure S1E). Typically, b2-b3 loops are <10 residues (Bousquet-Antonelli and Deragon, 2009), and a b2-b3 loop of this size is unprecedented to our knowledge. The insertion of a 47 residue b2-b3 loop provides a structural explanation for why an RRM fold was not predicted for the p65 C-terminal domain. Intriguingly, based on the structure and a DALI search (Holm and Rosenstrom, 2010) using p65-C2DL1 as a probe, the overall fold of p65 C-terminal domain is most similar to human La protein (hLa) RRM2 (Figure S2A) (Jacks et al., 2003). Based on the solution structure and NMR data, the remaining flexible residues of the b2-b3 loop and five N-terminal residues were deleted to make construct p65-C1DL2 (375–542D413– 459), which yielded crystals that diffracted to 2.5 A˚ (Figure 1A, Figure S2B, and Table S2). This protein construct retains the 22 C-terminal residues deleted in the NMR solution structure. The crystal structure (Figure 1F) is the same as the solution struc18 Molecular Cell 47, 16–26, July 13, 2012 ª2012 Elsevier Inc.

TER Binding by p65-C Requires the C-Terminal Tail but Not the b2-b3 Loop Since the binding site for p65-C includes the region of TER stem IV encompassing the GA bulge (O’Connor and Collins, 2006), we designed a minimal stem IV hairpin (S4) containing four base pairs on either side of the GA bulge and capped by a UUCG tetraloop (Figure 1B). In TER, the proximal stem IV contains a bulge U three base pairs from the GA bulge, but deletion of this nonconserved bulge U has no effect on activity (Richards et al., 2006). Gel mobility shift assays of p65C1 with S4, a longer stem IV construct, and stem-loop IV all gave comparable results (Figures S3A–S3D). Gel mobility shift assays did not show any interaction between p65-C1 and two control RNAs in which the GA bulge was deleted (S4-DGA) or replaced by UU (S4-UU) (Figures S3E and S3F). NMR titration of S4 or longer RNAs into a sample of p65-C1 resulted in shifting and/ or broadening of many of the crosspeaks in the 1H-15N HSQC spectra, indicating formation of a complex in slow-to-intermediate exchange on the NMR time scale (Figures S4A–S4C). These results indicate that our minimal stem IV is sufficient for specific binding of p65-C1. Stoichiometric gel mobility shift and NMR titration experiments show that truncation of the b2-b3 loop does not significantly affect RNA binding to S4 and binding is specific for the GA bulge (Figures S4D–S4F). In contrast, deletion of the C-terminal tail in p65-C2 or p65C2DL1 abolished both the gel shift and S4 interaction as judged by NMR (Figures S3G, S3H, S4G, and S4H). To quantify the strength of the interaction between p65-C and S4, equilibrium dissociation constant (KD) values were measured using isothermal titration calorimetry (ITC) (Figure 2, Figure S5, and Table 1). The p65-C1:S4 and p65-C1DL1:S4 complexes have comparable KDs of 36.4 ± 3.6 nM and 50.6 ± 7.1 nM, respectively (Figures 2A and 2B). This confirms that partial deletion of the b2-b3 loop has little if any impact on RNA binding.

Molecular Cell Structure of p65 xRRM2 Complex with Telomerase RNA

Table 1. Equilibrium Dissociation Constants and Stoichiometry Constant Values Determined Using ITC Measurements for p65-C Constructs with TER Stem IV Constructs Protein

RNA

p65-C1

S4

36.4 ± 3.6

1.1 ± 0.01

p65-C1DL1

S4

50.3 ± 7.1

1.2 ± 0.01

p65-C1DL2

S4

101 ± 21.2

1.2 ± 0.02

p65-C1

S4-UU

1,736 ± 153

1.4 ± 0.03c

p65-C1DL1

S4-UU

2,551 ± 156

1.4 ± 0.04c

p65-C1DL2

S4-UU

1,311 ± 120

1.9 ± 0.04c

p65-C1

S4-DGA

2,985 ± 220

1.5 ± 0.04c

p65-C1DL1

S4-DGA

2,340 ± 190

1.7 ± 0.04c

p65-C1DL2

S4-DGA

2433 ± 432

1.8 ± 0.04c

p65-C2

S4

KD (nM)

N

n.b.

a

n.a.b

n.b.

a

n.a.b

p65-C2DL1

S4

p65-T

S4

926 ± 220

p65-C1DL2 Y407A

S4

374.5 ± 26.6

1.2 ± 0.02

p65-C1DL2 R465A

S4

638.6 ± 46.8

1.3 ± 0.01c

1.8c

a

No binding. b Not applicable. c Significant deviations from a stoichiometry of 1:1 were only observed for weak binding complexes, consistent with a decrease in binding specificity.

Complete deletion of the b2-b3 loop in p65-C1DL2, which yielded crystals, has a small effect on RNA binding, decreasing the affinity <3-fold (Figure 2C). Binding affinities for interactions between p65-C1, p65-C1DL1, and p65-C1DL2 and S4-UU or S4-DGA were 50- to 80-fold lower than the same proteins and S4 (Table 1 and Figures S5A–S5F). No interaction was detected between p65-C2 or p65-C2DL1 and RNA by ITC, indicating that the KD must be >1 mM (Figure 2D and Figure S5G). These results confirm that specific interaction of p65 C-terminal domain with RNA requires a GA bulge flanked by helices, that the b2-b3 loop does not play any apparent role in binding to TER stem IV, and that the p65 C-terminal 22 residues are essential for high-affinity RNA binding. RNA interaction with the isolated C-terminal tail residues 515–542 (p65-T) is 30 fold weaker than by p65-C1 (Figure S5H and Table 1), indicating that highaffinity binding requires both the atypical RRM and the C-terminal tail. Crystal Structure of p65-C with Stem IV of TER A complex of p65-C1DL2 with S4 was crystallized, and the structure was solved to 2.6 A˚ (Figure 3A, Figure S6, and Table S2). Although S4 is a hairpin in solution, it crystallizes as a dimer, forming two symmetrical binding sites for protein (Figures S6A and S6B). Dimerization of the RNA retains the native binding site, with the nonnative UUCG hairpin loop paired as a duplex in the center, outside the binding site. In the complex, the C-terminal tail that is unstructured in the free protein forms an a-helical extension of helix a3 (hereafter called a3x), resulting in a single long C-terminal a helix with clear electron density visible up to residue 531 (Figure 3A). This helical extension binds across the major groove of S4 in the region corresponding to the top of proximal stem IV and wedges open the C-G base pairs on

either side of the GA bulge, resulting in a 105
bend in the RNA (Figures 3B and 3C). Both bases of the GA bulge are extruded out of the helix and interact with residues on the RRM (Figure 3A). Insertion of an a helix into the major groove of RNA at an internal loop was first observed for the Rev peptide (Battiste et al., 1996), but without the significant bend of the RNA helix that arises from the combination of the single- and double-strand interactions with the p65 RRM2 b sheet and helix a3x. The bulge Gua121 is inserted into the interface between residues on the b sheet and helix a3. A remarkable number of hydrophobic and hydrogen bond interactions stabilize Gua121 in this binding pocket (Figures 3B and 3D). The N1 and N2 protons on the Watson-Crick face are hydrogen bonded to the side chain of Asp409 on strand b2, and the N7 and O6 on the Hoogsteen face are hydrogen bonded to Arg465 side chain on strand b3. Tyr407 on strand b2 stacks directly below the Gua121 base, and the aliphatic part of the helix a3 Lys517 side-chain stacks above it. The Tyr407 hydroxyl group is within hydrogen bond distance of both the phosphate O2 and the Gua121 ribose 20 OH. The bulge Ade122 base is perpendicular to both Gua121 and Tyr407, and the side chain of Gln467 at the beginning of the b3-a2 loop stacks on the other side of Ade122. In contrast to Gua121, Ade122 is not sandwiched between helix a3 and the b sheet, but rather is located on the region of the b sheet above helix a3. Remarkably, the same Arg465 that recognizes the Hoogsteen face of Gua121 also recognizes the Hoogsteen face of the bulge Ade121, with its backbone carbonyl oxygen hydrogen bonded to Ade121 N6 and its side-chain amino hydrogen bonding to Ade121 N7 (Figure 3D). Helix a3x that binds across the major groove at the top of proximal stem IV is not linear with the rest of helix a3, but rather bends 12
where it contacts the dsRNA (Figure 3A). Stacking of the conserved C-G base pairs on either side of the GA bulge is completely disrupted, and these base pairs are rolled open toward the major groove. Three aromatic residues on helix a3 are inserted between these base pairs; the aromatic rings of Phe521 and Phe524 are perpendicular to the base planes, wedging them open, while Phe525 stacks directly on Gua147, below and perpendicular to Phe524 (Figures 3B and 3E). The interaction of helix a3x with the dsRNA is further stabilized by other side-chain interactions with phosphates of Gua146, Gua147, and Cyt120. Structural Changes in the p65 C-Terminal Domain upon Telomerase RNA Binding Except for the formation of the extended helix a3 on RNA binding from a disordered tail in the free protein, there are only small changes in the rest of the RRM (Figure 4A). Compared to the free protein crystal structure, in the complex helix a3 moves slightly away from the b sheet surface to accommodate the GA bulge, and strands b2 and b3 are extended by one residue each and show a small change in orientation to one that is similar to the solution structure of the free protein. Among the residues from strands b2 and b3 that interact with the GA bulge, only Tyr407 moves significantly, flipping down to stack on Gua121 (Figure 4A). This suggests that the binding pocket for the GA bulge (Figure 4B) is preformed in the free protein and that Tyr407 locks the RNA in place. Molecular Cell 47, 16–26, July 13, 2012 ª2012 Elsevier Inc. 19

Molecular Cell Structure of p65 xRRM2 Complex with Telomerase RNA

Figure 3. Crystal Structure of C-Terminal Domain with TER Stem IV

p65

(A) Two views of the p65-C1DL2:S4 complex. (B) Schematic of the protein-RNA interactions. (C) Stick rendering of the RNA on the surface of the protein, illustrating the 105
bend induced by protein binding. (D) Protein interactions with the GA bulge. (E) View of helix a3x in the major groove. Aromatic side chains that wedge open the base pairs adjacent to the GA bulge and other residues that interact with the RNA are labeled. Protein color scheme is as in Figure 1. Gua, Ade, and Cyt that contact the protein are green, magenta, and cyan, respectively, and the rest of the RNA is light blue.

extensively with helix a3x and may be important for stabilizing the ends of the two helices. Single-molecule FRET studies revealed that p65 binding to stem-loop IV brings stem I and loop IV closer together and that this compaction is dependent on the GA bulge (Robart et al., 2010; Stone et al., 2007). The structure of p65-C in complex with stem IV reveals the structural basis and details of this conformational change.

A Dramatic Change in the Telomerase RNA Structure upon p65 C-Terminal Domain Binding In contrast to the small changes in the protein structure, the RNA undergoes a large conformational change upon binding to protein. The central GA bulge and the G-C base pairs that flank the bulge are absolutely conserved in Tetrahymena ciliates. In the solution structure of stem-loop IV, Ade122 of the GA bulge is stacked into the helix, and the base of Gua121 is flipped out. The flanking base pairs are tilted open toward the GA bulge, resulting in an 43
bend between the proximal and distal helices (Chen et al., 2006) (Figure 4C). In the complex with p65, the Gua121 and Ade122 nucleotides are completely flipped out of the helix, widening the major groove to accommodate helix a3x, which induces a large roll between the flanking base pairs, opening toward the major groove and compressing the minor groove (Figure 4D). This conformational change in TER results in the 105
bend which could bring loop IV and the stem II spatially closer, facilitating TERT binding by bridging two TERT binding sites, i.e., loop IV and the TBE (Robart et al., 2010; Stone et al., 2007). The G-C base pairs that flank the GA bulge interact 20 Molecular Cell 47, 16–26, July 13, 2012 ª2012 Elsevier Inc.

Mutations of Residues that Recognize the GA Bulge Decrease TER Binding As discussed above, deletion of the C-terminal tail from p65 C-terminal domain results in complete loss of TER binding, showing that helix a3x is critical. In order to test the importance of residues from the b sheet in GA bulge recognition and binding, we generated single alanine point mutants of two key residues, Y407A (on b2) and R465A (on b3) in p65-C1DL2. Substitution of Y407 with alanine would disrupt the stacking interactions of the aromatic ring on Gua121 and dipole-dipole interactions with Ade122 in the GA bulge. ITC titrations results showed that p65-C1DL2 Y407A binds S4 with KDs of 374.5 ± 26.6 nM, which is approximately four times lower compared to p65-C1DL2 (Figure 2E and Table 1). Substitution of R465 with alanine would disrupt hydrogen bonds to both Gua121 and Ade122, and consistent with this, p65-C1DL2 R465A binds S4 more than six times more weakly (KD 638.6 ± 46.8 nM) compared to p65-C1DL2 (Figure 2F and Table 1). These results substantiate the importance of the interactions of these residues for RNA binding revealed in the crystal structure. Hierarchical Assembly of the Catalytic Core Requires the C-Terminal Tail but Not the b2-b3 Loop Since p65 also has N-terminal, La motif, and RRM1 domains that interact with other regions of TER, i.e., stem I and the 30 end containing the AUUUU-30 sequence (O’Connor and Collins, 2006;

Molecular Cell Structure of p65 xRRM2 Complex with Telomerase RNA

Figure 4. Comparison of Free and Bound Protein and RNA (A) Superposition of p65-C1DL2 free (green) and in complex with S4 (red). Side chains that form the GA bulge-binding pocket are shown. (B) The GA bulge residues in the protein-binding pocket. (C and D) Structures of the GA bulge and adjacent C-G base pairs in the (C) free RNA (PDB ID 2FEY) and (D) bound RNA, and schematics of the bend size and angle. RNA bend angles (tilt and roll) were measured using the program CURVES 5.1 (Lavery and Sklenar, 1988). Helix D and helix P represent the proximal and distal stem IV, respectively (see Figure 6C).

Prathapam et al., 2005), we investigated any potential influence of the C-terminal tail (a3x) or the b2-b3 loop on TER binding and hierarchical assembly in the context of full-length p65 (p65-FL) using electrophoretic mobility shift assays (EMSAs). The b2-b3 loop points away from the RNA in the complex (Figure 3A) but could potentially interact with regions of TER outside the minimal binding site. Partial deletion of the b2-b3 loop in p65-FLDL1 (D421–442) did not affect TER binding (KD 0.7 nM versus 0.6 nM for p65-FL), and complete deletion of loop residues in p65-FLDL2 (D413–459) decreased the binding by 3-fold (KD 2.0 nM) (Figures 5A–5C). These results are consistent with the results obtained for the interaction of p65 C-terminal domain constructs with S4 (Figures 2A–2C). Therefore, the 3fold decrease in binding of p65-FLDL2 with TER or p65-C1DL2 with S4 compared to that of wild-type p65-FL or p65-C, respectively, can be attributed to the fact that complete deletion of the loop results in a small change in position of the b2 and b3 strands in the free RRM2 rather than the loop playing a role in TER binding. Taken together, these results indicate that the b2-b3 loop does not play any detectable role in interaction with TER. In contrast, deletion of C-terminal tail residues 520–542 (p65-FLDT) results in reduction of binding affinity by >30-fold (KD 20 nM) (Figure 5D). The p65-TER complex stimulates the binding of TERT(1–516) to TER (O’Connor and Collins, 2006; Stone et al., 2007). We investigated this hierarchical assembly of p65-TER with TERT by EMSA (O’Connor and Collins, 2006) (Figures 5E–5H). Both p65-FLDL1:TER and p65-FLDL2:TER stimulate TERT binding to the same extent as p65-FL:TER complex (Figures 5E–5G). Thus, deletion of the b2-b3 loop has not only little effect on TER binding but also no effect on hierarchical RNP assembly, indicating that its role, if any, likely involves protein-protein inter-

actions in the context of the holoenzyme. In striking contrast, no ternary complex of TERT with p65-FLDT:TER is observed, indicating that p65-FLDT:TER does not stimulate TERT binding (Figure 5H). We note that deletion of the tail residues results in an altered mobility of the TER:p65 complex, likely due to a difference in TER conformation and/or specificity of binding. Previous studies had shown that p65 C-terminal domain was necessary and sufficient for hierarchal assembly (O’Connor and Collins, 2006; Prathapam et al., 2005). Here we show that deletion of the C-terminal tail (helix a3x) alone abolishes hierarchal assembly, even in the context of the rest of p65. Together with the structure of the p65-C1DL2:S4 complex, these results show that cooperative binding of the C-terminal domain b sheet and helix a3x to the region of TER stem-loop IV containing the GA bulge is required for assembly of the catalytic core. xRRM, a New RRM Specific to Genuine La and LARP7 Proteins Tetrahymena telomerase p65 and Euplotes telomerase p43 are members of a family of La-related proteins 7 (LARP7) (Bayfield et al., 2010; Bousquet-Antonelli and Deragon, 2009), which also includes the human 7SK binding protein hLARP7 (also called PIP7S and HDCMA18) (Diribarne and Bensaude, 2009; He et al., 2008). Genuine La proteins bind to a 30 -terminal oligo(U) tract of three or more nucleotides (UUU-30 OH) (Alfano et al., 2004; Bayfield et al., 2010; Bousquet-Antonelli and Deragon, 2009; Kotik-Kogan et al., 2008; Teplova et al., 2006; Wolin and Cedervall, 2002), without other known sequence specificity, while the LARP7 family has stronger specificity for other RNA sequences in addition to recognizing the UUU-30 OH. The predicted domain structure of the LARP7 family is similar to genuine La proteins, in that all proteins contain a LAM followed by an RRM1 and usually an RRM2. For p65, only the LAM and RRM1 domains were predicted (Bayfield et al., 2010; Bousquet-Antonelli and Deragon, 2009). We aligned the sequences of p65 C-terminal domain with the sequences of 33 LARP7 and genuine La family proteins (Figure 6A and Figure S7). The alignment predicts a common Molecular Cell 47, 16–26, July 13, 2012 ª2012 Elsevier Inc. 21

Molecular Cell Structure of p65 xRRM2 Complex with Telomerase RNA

Figure 5. Hierarchical Assembly of p65, TERT, and TER (A–D) EMSA of TER with various p65-FL variants: (A) p65-FL, (B) p65-FLDL1, (C), p65-FLDL2, and (D) p65-DT. The bands corresponding to the migration of free TER and TER-p65 complexes are marked on right. At higher concentrations of p65-FL, p65-FLDL1, and p65-FLDL2, a second band corresponding to a supershifted p65-TER higher-order complex appears (O’Connor and Collins, 2006). (E–H) Stimulation of TERT binding to TER in the presence of p65-FL variants: (E) p65-FL, (F) p65-FLDL1, (G) p65-FLDL2, and (H) p65-FLDT. For each assay, TERTTER interaction was assayed on the same gel (lanes 1–5). TERT (1–516) concentration required to shift half of the p65-TER complexes is denoted using an asterisk. No stimulation of TERT interaction for TER was observed for p65-FLDT-TER (H). The key identifying the various bands in (E)–(H) is shown at the right of (H).

atypical RRM with the features found in the structures of p65 (except for the long b2-b3 loop) and hLa protein RRM2 (Jacks et al., 2003), the only other member of this family for which structural data are available, including a C-terminal extension that is unstructured in the free protein. We note that Euplotes p43 does not have a long b2-b3 loop, consistent with the absence of a role for this loop in Tetrahymena telomerase core RNP assembly. The sequence alignment also reveals that the b2 residues that are involved in recognition of Gua121 in the GA bulge are conserved in LARP7 and genuine La proteins, forming a new Aromatic-X-D/E/Q/N motif that we define as RNP3 (Figure 6A). The Arg465 on the nonconsensus RNP1 on b3, which recognizes both nucleotides in the GA bulge, is also highly conserved (Figure 6A and Figure S7). The decrease in TER binding affinities of p65-C1DL2 mutants Y407A (corresponding to the aromatic residue of RNP3 motif) and R465A supports the importance of these residues in RNA interactions (Figures 2E and 2F and Table 1). Similar to RNP1 and RNP2 on the canonical RRMs, RNP3 could recognize diverse nucleotides (Allain et al., 2000; Clery et al., 2008). The other families of LARP (i.e., LARP1, LARP4, and LARP6) (Bayfield et al., 2010; Bousquet-Antonelli and Deragon, 2009) do not share these conserved features in RRM2. In addition, the p65 C-terminal domain structure and biochemical studies reported here reveal that one of the conserved features of genuine La proteins, an intrinsically disordered C terminus (Alfano et al., 2004; Dong et al., 2004; Jacks et al., 2003; Kucera et al., 2011), is common to the LARP7 family 22 Molecular Cell 47, 16–26, July 13, 2012 ª2012 Elsevier Inc.

as well (Figures 6A and 6B). We suggest naming this domain xRRM for atypical RRM with C-terminal extension and identifying the recognition motif on b2 as RNP3. Genuine La proteins bind the 30 UUUOH of newly synthesized Pol III and some Pol II noncoding RNA transcripts with the LAM and RRM1 domains, protecting them from degradation (Bayfield and Maraia, 2009; Kotik-Kogan et al., 2008; Teplova et al., 2006; Wolin and Cedervall, 2002). La proteins also function as RNA chaperones for correct folding of diverse RNAs such as some tRNAs, snRNAs, snoRNAs, and mRNA structural elements (Kucera et al., 2011; Wolin and Cedervall, 2002). The C-terminal region of genuine La proteins is highly divergent, with limited sequence similarity. In human La protein, the C-terminal tail harbors several functionally important elements such as phosphorylation sites, nuclear localization signal (NLS), and nuclear retention element (NRE) (Bayfield et al., 2010). Several studies have implicated RRM2 and the C-terminal tail of human La protein as important for cellular mRNA translation and critical for HCV virus RNA translation (Costa-Mattioli et al., 2004), but identification of a role for RNA binding by the La RRM2 has been elusive, as exemplified by the structural study of Jacks et al. (Jacks et al., 2003), which failed to show RNA binding. Because the helix a3 occludes the region on the b sheet surface where single-strand nucleotides usually bind, it has been assumed that single-strand nucleotides would not bind on the b sheet (Jacks et al., 2003). Very recently it has been reported that RRM2 of human La cooperates with LAM and RRM1 to

Molecular Cell Structure of p65 xRRM2 Complex with Telomerase RNA

Figure 6. La and LARP7 Proteins Have a Common RRM2 Structure (A) Sequence alignment of the C-terminal region of three LARP7 family proteins, Tetrahymena telomerase p65 (Tt-p65), Euplotes telomerase p43 (Ea-p43), and human 7SK protein hLARP7 (Hs-LARP7); and human genuine La family protein, hLa (Hs-La). Secondary structure elements of p65 and hLa protein are shown above (orange and red) and below (blue) the sequence, respectively. The nonconsensus RNP1 and RNP2 are boxed in green with positions where conserved aromatic residues in typical RNPs would be marked with an asterisk. A conserved RNA binding motif on b2 identified in this study is labeled RNP3 and boxed in red. The red asterisks are conserved residues in RNP3, which in p65 xRRM2 interact with RNA. (B) Domain architecture of p65, p43, hLARP7, and hLa. (C) Model of the interaction of the LAM, RRM1, and xRRM2 domains of p65 on stem-loop IV. LAM and RRM1 domains are based on homology modeling with the crystal structure of hLa LAM and RRM1 complex with UUUA (PDB ID 2VOP), and loop IV is from PDB ID 2H2X. A schematic of the interactions is shown on the right. For description of the model building, see the Supplemental Experimental Procedures.

interact with IRES domain IV of hepatitis C virus (HCV) RNA (Martino et al., 2012). In S. cerevisiae La, Lhp1p, the C terminus (which follows its single RRM) is required for accumulation and/or correct folding of certain noncoding RNA precursors, and there is evidence that it becomes structured on binding RNA targets (Kucera et al., 2011). Our results unambiguously show that an xRRM can bind RNA specifically with high affinity. We propose that the homologous xRRM2s in other LARP7 and genuine La proteins interact with structured RNAs in a similar manner to p65, with the atypical RRM providing a binding site for nucleotides in loops and the intrinsically disordered C-terminal tail forming a helix (a3x) that binds the major groove of dsRNA near the loop. Thus the structure of the p65 C-terminal domain-TER complex reveals a new mode for RNA recognition combining ssRNA and dsRNA binding by a new class of RRM that appears to be unique to genuine La and LARP7 proteins. This mode of binding may be general to the chaperone function of genuine La and LARP7 proteins, potentially explaining how xRRM2 would function in the biogenesis of various RNAs (Alfano

et al., 2004; Dong et al., 2004; Jacks et al., 2003; Kucera et al., 2011; Martino et al., 2012). Model of Assembly of p65 with TER The structure of p65 xRRM2 in complex with RNA is the first structure of a telomerase-specific protein in complex with TER and explains why this domain is sufficient to direct hierarchical assembly of the catalytic core in vitro, as it induces an essential conformational change in stem IV required for efficient TERT binding. Based on our present study and the structural homology of p65 with genuine La and LARP7 family proteins, we propose that the p65 LAM and RRM1 domains could function in the early steps of telomerase holoenzyme biogenesis in a manner similar to that of genuine La proteins, cooperatively binding to TER UUU30 OH after it is transcribed by Pol III. Since the Tetrahymena TER UUU30 OH is not subsequently degraded, the LAM+RRM1 would remain associated with TER, positioning xRRM2 to bind to the adjacent proximal stem-loop IV and GA bulge, as modeled in Figure 6C. In the folded secondary structure of ciliate TERs, Molecular Cell 47, 16–26, July 13, 2012 ª2012 Elsevier Inc. 23

Molecular Cell Structure of p65 xRRM2 Complex with Telomerase RNA

stem I brings the 50 end of TER close to the 30 end. A model consistent with the accumulated data is that the N-terminal domain of p65 binds to the 50 end and/or stem I of TER and the LAM and RRM1 bind to the 30 end, to help correctly fold stem I and stem-loop IV. Thus, p65 plays at least three essential roles in telomerase biogenesis: first, it protects the 30 end of TER from degradation; second, it acts as a chaperone to correctly fold TER for protein binding; and third, it bends TER stem-loop IV to position it for interaction of loop IV with TERT RNA binding domain. EXPERIMENTAL PROCEDURES Protein and RNA Preparation p65 constructs used in these studies were cloned and expressed in E. coli and purified by standard methods as described in the Supplemental Experimental Procedures. TERT(1–516) was purified as described (O’Connor and Collins, 2006; Stone et al., 2007). RNAs used in this study were prepared by in vitro transcription and purified as described in the Supplemental Experimental Procedures. NMR Spectroscopy and Structure Calculations NMR protein samples were concentrated to 0.8–1.2 mM in 20 mM NaH2PO4 (pH 7.0), containing 50 mM NaCl, 1 mM 1,4-DL-dithiothreitol, and 0.02% NaN3 (in 90% H2O/10% D2O). NMR experiments were performed at 298 K on 800 and 500 MHz spectrometers equipped with HCN cryoprobes. Backbone and side-chain assignments of p65-C2 and p65-C2DL1 were obtained using a combination of standard triple resonance experiments (Cavanagh et al., 2006). Further details on NMR data acquisition, including RDCs and heteronuclear NOE measurements, and resonance and NOE assignments are given in the Supplemental Experimental Procedures. The RNA binding to the various constructs of p65 C-terminal domain was characterized by monitoring chemical shift changes in 1H-15N HSQC spectra of 0.2 mM 15N-labeled protein when various TER RNAs were added stepwise up to 0.6 or 0.8 mM (Figure S4). The structure of p65-C2DL1 was calculated in a semiautomated iterative manner with the program CYANA 2.1 (Guntert, 2004). A few manually assigned NOEs and chemical shift assignments were supplied to CYANA 2.1, and using 100 starting conformers, CYANA 2.1 protocol was applied to calibrate and assign further NOE crosspeaks. Dihedral angle restraints were generated using TALOS+ and were also added to the calculation (Shen et al., 2009). After the first few rounds of automatic calculations, the NOESY spectra were analyzed again to identify additional crosspeaks consistent with the structural model and to correct misidentified NOEs. Slowly exchanging amides involved in hydrogen bonds were identified by redissolving the lyophilized protein in 100% 2H2O and collecting 1H-15N HSQC spectra at various time intervals. Hydrogen-bond constraints were then added to the structure calculation protocol. The distance constraints from the 20 conformers with lowest target energy functions (final average target function 3.12 ± 0.24) were converted to XPLOR format using h20refine.py script (Jung et al., 2005; Linge et al., 2003). The final 200 structures were calculated in XPLOR following a standard protocol with incorporation of RDCs. Structures were validated by PROCHECK-NMR (Laskowski et al., 1996) and visualized using PyMol (http://www.pymol.org/). The structural statistics are presented in Table S1. Crystallization and Crystal Structure Calculations Free p65-C1DL2 and p65-C1DL2:S4 were crystallized using the hanging drop, vapor diffusion method. Details of the crystallization conditions are described in the Supplemental Experimental Procedures. Unit cell dimensions and other parameters are given in Table S2. The p65-C1DL2:S4 data sets were collected at the Advanced Photon Source (APS) at beamline 24-ID-C (NE-CAT). Data sets were collected at three different wavelengths for peak, inflection, and high remote data sets (Table S2). Data were indexed, integrated, and scaled using XDS (Kabsch, 2010). Macromolecular phasing was carried out by HKL2MAP (Pape and Schneider, 2004) using the multiple anomalous disper-

24 Molecular Cell 47, 16–26, July 13, 2012 ª2012 Elsevier Inc.

sion (MAD) method. The resulting phases were improved by using the DM program (CCP4, 1994) and used for manual model building in Coot (Adams et al., 2010; Emsley and Cowtan, 2004). Final iterative rounds of model building and refinement were carried out using Coot and PHENIX (Emsley and Cowtan, 2004) with TLS refinement (Painter and Merritt, 2006). Final data collection, phasing, and refinement statistics are presented in Table S2. There are two molecules of protein bound to a duplex RNA in the asymmetric unit. The RNA in the crystal forms a duplex with two identical halves, with one protein bound to each half. There is clear electron density for all of the RNA nucleotides and for the protein except for the initial His-tag containing eight to nine residues and C-terminal residues (532–542). The data set for p65-C1DL2 was collected at 2.5 A˚ at APS beam line 23-ID-D (GM/CA). Data were indexed and integrated using Mosflm (Leslie, 1992). The structure of the free protein was solved by molecular replacement with the protein structure in the p65-C1DL2:S4 complex using Phaser and Molrep programs in CCP4 suite (CCP4, 1994; McCoy et al., 2007; Potterton et al., 2003; Vagin and Teplyakov, 1997). The resulting model was inspected and finished manually with the program Coot (Emsley and Cowtan, 2004). Final iterative rounds of model building and refinement were carried out using Coot and PHENIX (Emsley and Cowtan, 2004; Adams et al., 2010). Final data collection, phasing, and refinement statistics are presented in Table S1. The asymmetric unit contains six molecules of protein. All molecules are similar, with the few differences located mostly in the C-terminal region and flexible region from residues 480 to 488. Most of the models have clear electron density for all residues, except for the N-terminal his-tag (ten residues) and C-terminal residues from 520 to 542. These parts are not discussed in the interpretation of results. Additionally, some side chains with no interpretable electron density were omitted in the final model. Isothermal Calorimetry Titration Binding of various p65 C-terminal domain constructs to RNA was measured by ITC using a MicroCal Omega VP-ITC system (MicroCal, Northampton, MA). Both RNA and proteins were extensively dialyzed against the NMR buffer. The proteins at concentrations between 150 and 200 mM were titrated into 5–10 mM RNA at 298 K. Calorimetric data were analyzed with ORIGIN V5.0 (MicroCal). The binding parameters DH, association constant K, and n were kept floating during the fits. The average heat change from the last four injections was subtracted from the data to account for the heat of dilution. Electrophoretic Mobility Shift and Catalytic Core Assembly Assays The EMSA and catalytic core assembly assays were done essentially as described previously (O’Connor and Collins, 2006), with minor modifications. Radiolabeled TER was synthesized in vitro using T7 polymerase and 32PUTP and gel purified. For EMSA, 20 ml binding reaction was assembled by mixing 0.1 nM ‘‘body-labeled’’ 32P-U-labeled RNA, 500 ng yeast tRNA (Sigma-Aldrich), 0.25 ml of RNAsin (Promega), 5 mM DTT, and protein dilutions in buffer (20 mM Tris base [pH 8], 1 mM MgCl2, 100 mM NaCl). The binding reaction was incubated at room temperature for 30 min followed by gel electrophoresis on a 6% (37.5:1) polyacryalamide gel at 4
C for 2.5 hr. For the catalytic core assembly assays, TER was incubated with various proteins for 30 min at room temperature followed by addition of TERT(1– 516). TERT(1–516) contains all of the primary sites of RNA-TERT interaction, but unlike the full-length TERT, it can be expressed in a soluble recombinant form (O’Connor and Collins, 2006; Stone et al., 2007). The concentration of proteins used is indicated on Figures 5A–5D. Control reactions with TER and TERT were run alongside on the same gel. ACCESSION NUMBERS Coordinates and restraints for the 20 lowest energy structures of p65 C-terminal domain (p65-C2DL1) and coordinates and structure factors for p65 C-terminal domain (p65-C1DL2) and p65 C-terminal domain in complex with stem IV RNA (p65-C1DL2:S4) have been deposited in the Protein Data Bank under accession numbers 2LSL, 4EYT, and 4ERD, respectively. Chemical shifts for p65-C2DL1 have been deposited in the BioMagResBank, accession number 18435.

Molecular Cell Structure of p65 xRRM2 Complex with Telomerase RNA

SUPPLEMENTAL INFORMATION

(TER) and rescues telomerase reverse transcriptase and TER assembly mutants. Mol. Cell. Biol. 30, 4965–4976.

Supplemental Information includes seven figures, two tables, Supplemental Experimental Procedures, and Supplemental References and can be found with this article online at doi:10.1016/j.molcel.2012.05.018.

Bertuch, A.A., and Lundblad, V. (2006). The maintenance and masking of chromosome termini. Curr. Opin. Cell Biol. 18, 247–253.

ACKNOWLEDGMENTS This work was supported by NSF and NIH grants to J.F. and by NIH grant to K.C. The authors thank Drs. C.J. Park, R. Peterson, and Q. Zhang for help with NMR; K. Stahmer for help in the early stages of this work; M. Zhao for help with X-ray data collection; and the staff members of beamlines ID-24 (NE-CAT) and ID-23 (GM/CA) at APS of Argonne National Laboratory and CCP4 APS School 2011 for help with data collection and analysis. M.S. designed experiments, prepared samples, performed experiments, analyzed the data, and wrote the paper; Z.W. helped refine the NMR structure and performed sequence alignment; B.-K.K. helped with NMR structure calculations; A.P. prepared protein and RNA samples; D.C. helped with X-ray data collection and analysis; K.C. provided reagents and edited the paper; and J.F. designed experiments, analyzed data, supervised all aspects of the work, and wrote the paper. Received: December 26, 2011 Revised: March 2, 2012 Accepted: May 10, 2012 Published online: June 14, 2012 REFERENCES Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221. Aigner, S., Postberg, J., Lipps, H.J., and Cech, T.R. (2003). The Euplotes La motif protein p43 has properties of a telomerase-specific subunit. Biochemistry 42, 5736–5747. Akiyama, B.M., Loper, J., Najarro, K., and Stone, M.D. (2012). The C-terminal domain of Tetrahymena thermophila telomerase holoenzyme protein p65 induces multiple structural changes in telomerase RNA. RNA 18, 653–660. Alfano, C., Sanfelice, D., Babon, J., Kelly, G., Jacks, A., Curry, S., and Conte, M.R. (2004). Structural analysis of cooperative RNA binding by the La motif and central RRM domain of human La protein. Nat. Struct. Mol. Biol. 11, 323–329. Allain, F.H., Gubser, C.C., Howe, P.W., Nagai, K., Neuhaus, D., and Varani, G. (1996). Specificity of ribonucleoprotein interaction determined by RNA folding during complex formulation. Nature 380, 646–650. Allain, F.H., Bouvet, P., Dieckmann, T., and Feigon, J. (2000). Molecular basis of sequence-specific recognition of pre-ribosomal RNA by nucleolin. EMBO J. 19, 6870–6881. Artandi, S.E., and DePinho, R.A. (2010). Telomeres and telomerase in cancer. Carcinogenesis 31, 9–18. Autexier, C., and Lue, N.F. (2006). The structure and function of telomerase reverse transcriptase. Annu. Rev. Biochem. 75, 493–517. Battiste, J.L., Mao, H., Rao, N.S., Tan, R., Muhandiram, D.R., Kay, L.E., Frankel, A.D., and Williamson, J.R. (1996). Alpha helix-RNA major groove recognition in an HIV-1 rev peptide-RRE RNA complex. Science 273, 1547– 1551. Bayfield, M.A., and Maraia, R.J. (2009). Precursor-product discrimination by La protein during tRNA metabolism. Nat. Struct. Mol. Biol. 16, 430–437. Bayfield, M.A., Yang, R., and Maraia, R.J. (2010). Conserved and divergent features of the structure and function of La and La-related proteins (LARPs). Biochim. Biophys. Acta 1799, 365–378. Berman, A.J., Gooding, A.R., and Cech, T.R. (2010). Tetrahymena telomerase protein p65 induces conformational changes throughout telomerase RNA

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