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The AAA-ATPase NVL2 is a telomerase component essential for holoenzyme assembly
Biochemical and Biophysical Research Communications 417 (2012) 1086–1092
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
The AAA-ATPase NVL2 is a telomerase component essential for holoenzyme assembly Joonyoung Her, In Kwon Chung ⇑ Departments of Biology and Integrated Omics for Biomedical Science, Yonsei University, Seoul 120-749, South Korea
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Article history: Received 19 December 2011 Available online 27 December 2011 Keywords: Telomerase hTERT NVL2 AAA-ATPase Nucleolus
a b s t r a c t Continued cell proliferation requires telomerase to maintain functional telomeres that are essential for chromosome integrity. Although the core enzyme includes a telomerase reverse transcriptase (TERT) and a telomerase RNA component (TERC), a number of auxiliary proteins have been identified to regulate telomerase assembly, localization, and enzymatic activity. Here we describe the characterization of the AAA-ATPase NVL2 as a novel hTERT-interacting protein. NVL2 interacts and co-localizes with hTERT in the nucleolus. NLV2 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT. Depletion of endogenous NVL2 by small interfering RNA led to a decrease in hTERT without affecting the steady-state levels of hTERT mRNA, thereby reducing telomerase activity, suggesting that NVL2 is an essential component of the telomerase holoenzyme. We also found that ATP-binding activity of NVL2 is required for hTERT binding as well as telomerase assembly. Our findings suggest that NVL2, in addition to its role in ribosome biosynthesis, is essential for telomerase biogenesis and provides an alternative approach for inhibiting telomerase activity in cancer. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Telomeres are essential and functional components located at the physical ends of eukaryotic chromosomes and are responsible for maintaining chromosome stability [1]. In most eukaryotic organisms, telomeres consist of long tracts of duplex telomere repeats (TTAGGG in vertebrates) with 30 single-stranded G overhang [2] and proteins that associate directly and indirectly with telomeric DNA sequences [3,4]. In the absence of functional telomere maintenance pathways, dividing cells show a progressive loss of telomeric DNA during each round of cell division [5]. Although recombination-based telomere elongation has been demonstrated for replenishing telomeric DNA [6], the major mechanism to compensate for telomere loss involves ongoing elongation of telomeric repeats by telomerase [7,8]. Telomerase activity is observed in most tumor cells but undetectable in normal somatic cells [9], suggesting an attractive molecular target for anti-cancer therapy. Although the core enzyme includes a telomerase reverse transcriptase (TERT) and a telomerase RNA component (TERC) [10], a number of auxiliary proteins have been identified to regulate telomerase assembly [11,12], localization [13,14], and enzymatic activity [15,16].
⇑ Corresponding author. Address: Department of Integrated Omics for Biomedical Science, Yonsei University, 134 Shinchon-dong, Seoul 120-749, South Korea. Fax: +822 364 8660. E-mail address: [email protected] (I.K. Chung). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.12.101
In a search for proteins capable of interacting with human telomerase, we identified nuclear valosin-containing protein-like (NVL) as an hTERT-interacting partner in a yeast two-hybrid screen. NVL was first identified as a member of the chaperone-like AAA-ATPase (for ATPase associated with various cellular activities) family with two conserved ATP-binding domains, which displays a high level of amino acid similarity with a cytosolic chaperone, VCP/ p97 [17]. NVL exists as two alternatively spliced forms, NVL1 (a short form) and NVL2 (a long form), which are produced from different methionines as the translation initiation sites. Whereas NVL1 is present in the nucleoplasm, NVL2 is mainly localized in the nucleolus where it is involved in the biogenesis of the 60S ribosomal subunit [18]. In this process, NVL2 has been shown to associate with ribosomal protein L5 [18] and RNA helicase DOB1 [19]. More recently, NVL2 was reported to interact with nucleolin through its N-terminal unique domain and facilitate the recycling of nucleolin using its ATPase activity for efficient ribosome biogenesis [20]. Here we report the characterization of NVL2 as an hTERT-interacting protein. NVL2 physically interacts and co-localizes with hTERT in the nucleolus. NVL2 also associates with catalytically competent telomerase through an interaction with hTERT. Whereas overexpression of NVL2 has no effect on telomerase activity, depletion of endogenous NVL2 in telomerase-positive cells led to a decrease in hTERT without affecting the steady-state levels of hTERT mRNA, thereby reducing telomerase activity. Overall, our data suggest that NVL2, in addition to its participation in ribosome
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biogenesis, is required for telomerase assembly and has a unique and independent role in the regulation of telomerase activity. Hence, NVL2 represents an alternative pathway for modulating telomerase activity in cancer.
35,000 rpm for 15 h at 4 °C in a linear 10–30% (v/v) glycerol gradient in a Beckman SW41Ti rotor. Fractions of 0.5 ml each were collected from the bottom of the gradient, and 15 ll of each fraction was analyzed for immunoblots.
2. Materials and methods
2.6. RNA Interference
2.1. Cell culture and plasmids
The siRNA target sequences specific for NVL were 50 -GACUCGUUAGACCCUGCUU-30 for siNVL-1 and 50 -GGCAAGACAGAAGAG UGGAAAUGAA-30 for siNVL-2. The siRNA duplexes were transfected into HeLa cells using RNAiMax transfection reagent (Invitrogen). A scramble sequence (50 -AATCGCATAGCGTATGCCGTT-30 ) was used as a control that did not correspond to any known gene.
HeLa and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 lg/ml streptomycin in 5% CO2 at 37 °C. The 3 FLAG-hTERT expression vector was constructed by inserting the full-length hTERT cDNA into pCMV7.1 3 FLAG vector (Sigma), and the hTERT-HA expression vector has been described previously [13]. Mammalian expression plasmids for FLAG-tagged wild-type NVL2 and mutants (K311M and K628M) were kind gifts from Dr. Mitsuo Tagaya (Tokyo University of Pharmacy and Life Science, Japan) [19]. The K311,628M double point mutant was generated by site-direct mutagenesis. The NVL1-V5 and NVL2-V5 expression vectors were constructed by inserting the PCR fragments into pcDNA3.1/V5-His (Invitrogen). The expression vectors for GSTNVL2 were constructed by cloning the full-length and truncated proteins from the NVL2 cDNA into pGEX-4T-1 (GE Healthcare). 2.2. GST pull-down, immunoprecipitation, and immunoblot analyses GST pull-down and immunoprecipitation were performed as previously described [16]. Immunoprecipitation and immunoblot analyses were performed using anti-NVL (Abnova), anti-FLAG (Sigma), anti-HA (Santa Cruz Biotechnology), anti-V5 (Invitrogen), anti-Reptin (Santa Cruz Biotechnology), anti-pontin (Santa Cruz Biotechnology), anti-tubulin (Santa Cruz Biotechnology), and antiactin (Sigma) antibodies as specified. Immunoblotting experiments were repeated at least three times and a representative blot is shown. 2.3. Telomerase assay The telomeric repeat amplification protocol (TRAP) was used as previously described [21]. Briefly, cell extracts (200 ng of protein) were added to the telomerase extension reactions and incubated for 20 min at 37 °C, and PCR was performed using the HTS primer and HACX primer for 30 cycles. As an internal telomerase assay standard, NT and TSNT primers were added to the PCR mixture. 2.4. Immunofluorescence microscopy Cells were grown on glass coverslips fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min and permeabilized in 0.2% Triton X-100 in PBS for 20 min. Cells were then blocked in 5% bovine serum albumin and incubated with goat anti-hTERT (Santa Cruz Biotechnology), mouse anti-NVL (Abnova) or rabbit anti-nucleolin (Santa Cruz Biotechnology) antibodies overnight at 4 °C. After washing with PBS, cells were incubated with Alexa Fluor 488-labeled rabbit anti-goat or goat anti-rabbit immuoglobulin and Alexa Fluor 546-labeled goat anti-mouse immunoglobulin (Molecular Probes). DNA was stained with 4,6diamino-2-phenylindole (Vectashield, Vector Laboratories) for 1 h at room temperature. Immunofluorescence images were captured using a confocal laser-scanning microscope (Carl Zeiss). 2.5. Glycerol gradient sedimentation Glycerol gradient sedimentation experiments were performed as previously described [22]. Briefly, extracts were centrifuged at
3. Results 3.1. Identification of NVL2 as an hTERT-interacting partner To identify hTERT-interacting factors, we screened a HeLa cell cDNA library using the yeast two-hybrid system [16]. With the carboxy-terminal domain of hTERT (amino acids 946-1132) as bait, a clone containing the full-length NVL2 cDNA was obtained and sequenced. NVL was first identified as a gene product that displays a high level of amino acid similarity with an AAA-ATPase, VCP/p97 [17] and has two alternatively spliced isoforms. Because NLV2 is exclusively localized in the nucleolus where the telomerase ribonucleoprotein (RNP) complex is assembled, we are particularly interested in identification of a nucleolar protein that can associate with hTERT. To confirm the direct interaction between hTERT and NVL2, we performed GST pulldown experiments. GST-NLV2, but not GSThRAP1 or the control GST, precipitated hTERT-HA expressed in HeLa cells, indicating that NVL2 interacts with hTERT in vitro (Fig. 1A). To determine whether hTERT and NVL2 associate in vivo, HeLa cells were co-transfected with hTERT-HA and NVL2Flag expression vectors and subjected to immunoprecipitation. hTERT-HA was detected in anti-Flag immunoprecipitates when NVL2-Flag was expressed (Fig. 1B). Likewise, NVL2-Flag was recovered in anti-HA immunoprecipitates when hTERT-HA was expressed. Endogenous NVL2 was also immunoprecipitated by hTERT-HA (Fig. 1C), indicating that NVL2 interacts with hTERT in mammalian cells. To map the region in NVL2 that is important for hTERT binding, a series of NVL2 fragments were fused to GST and used in the in vitro binding assay (Fig. 1D). Full-length NVL1 and NVL2 were found to associate with hTERT while other truncated forms failed to interact with hTERT (Fig. 1E). NVL2 has much higher affinity for hTERT binding than its shorter form, NVL1. We next determined the subcellular localization of NVL2 and hTERT by immunofluorescence staining. Endogenous NVL2 signals were almost exclusively detected in the nucleolus, as demonstrated by the finding that nucleolin clearly co-localized with NVL2 (Fig. 1F). Although some found in the nucleoplasm, hTERT was predominantly co-localized with NVL2 in the nucleolus (Fig. 1G). Given that telomerase ribonucleoprotein (RNP) is assembled in the nucleolus before translocating into the cajal body [23,24], the observations that NVL2 interacts and co-localizes with hTERT in the nucleolus suggest that NVL2 may participate in telomerase biogenesis. 3.2. NVL2 associates with catalytically competent telomerase Because NVL2 interacts with hTERT, we examined whether NVL2 associates with catalytically competent telomerase. HeLa cells transfected with NVL1-V5 or NVL2-V5 (or the empty vector) were subjected to immunoprecipitation with anti-V5 antibody and analyzed for telomerase activity by the telomerase repeat
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Fig. 1. NVL2 interacts and co-localizes with hTERT. (A) GST, GST-NVL2, or GST-hRAP1 were immobilized on glutathione-Sepharose and incubated with ectopically expressed hTERT-HA. Bound proteins were detected by immunoblotting with anti-HA antibody. (B) HeLa cells were co-transfected with hTERT-HA and NVL2-Flag and subjected to immunoprecipitation with anti-Flag or anti-HA antibodies, followed by immunoblotting as indicated. The asterisk marks the position of nonspecific proteins. (C) HeLa cells were transfected with hTERT-HA and subjected to immunoprecipitation with anti-HA antibody, followed by immunoblotting with anti-NVL2 antibody. (D) Schematic representation of NVL2 truncations fused to GST. The numbering of amino acids starts from the initiation methionine of NVL2. UD, NVL2 unique domain; AAA, AAA domain. (E) GST or the various truncated GST-NVL2 fusion proteins were affinity-purified and incubated with ectopically expressed hTERT-HA. The purified GST fusion proteins were visualized by Coomassie staining and indicated with arrowheads. Molecular mass markers are shown in kilodaltons. (F) HeLa cells were analyzed by immunofluorescence for co-localization of NVL2 with nucleolin. Immunofluorescence was used to detect endogenous NVL2 (red) and nucleolin (green). DNA was stained with 4,6-diamidino-2phenylindole (DAPI) (blue). (G) Immunofluorescence was used to detect endogenous NVL2 (red) and hTERT (green). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. NVL2 associates with catalytically competent telomerase. (A) HeLa cells transfected with NVL1-V5 or NVL2-V5 (or the empty vector) were subjected to immunoprecipitation with anti-V5 antibody and analyzed for telomerase activity by the TRAP assay. ITAS represents the internal telomerase assay standard. (B) Extracts from HEK293 cells were fractionated through 10–30% (v/v) glycerol gradients. Fractions were subjected to Western blotting to visualize NVL2, reptin, and pontin and analyzed for telomerase activity. (C) Extracts from HEK293 cells expressing Flag-hTERT were subjected to sedimentation analysis.
amplification protocol (TRAP) assay. Telomerase activity was detected in anti-V5 immunoprecipitates in cells expressing NVL1 and NVL2 but not in the control cells (Fig. 2A). The amount of telomerase activity recovered from NVL2-expressing cells was significantly higher than that recovered from NVL1-expressing cells even though the higher level of NVL1-V5 was expressed compared to NVL2-V5. This could be due to higher affinity of NVL2 for hTERT binding as shown in Fig. 1E. These results indicate that NVL2 associates with catalytically competent telomerase through an interaction with hTERT, and that this association could be specific. To further confirm that NVL2 associates with telomerase complex, extracts from HEK293 cells were subjected to sedimentation analysis on a linear 10–30% (v/v) glycerol gradient. Western blot analysis revealed that endogenous NVL2 was fractionated as a single peak spanning fractions 5–9 (Fig. 2B). Endogenous telomerase activity sedimented in fractions 1–9 with the peak centered on fraction number 5. Whereas telomerase activity cosedimented with NVL2 in fractions 5–9, NVL2 signal was not detected in fractions 1–3 (Fig. 2B). Because telomerase activity was immunoprecipitated by NVL2, these results suggest that catalytically competent telomerase exists in at least two different complexes; one complex contains NVL2 (fractions 5–7), and the other is free from NVL2 (fractions 1–3). We next wished to determine the sedimentation behavior of hTERT. However, we could not detect endogenous hTERT possibly due to its dilution in glycerol gradient fractions. Thus, HEK293 cells were transfected with Flag-hTERT and subjected to sedimentation analysis under identical conditions (Fig. 2C). Flag-hTERT sedimented in fractions 1–11 with the peak in fraction number 5, suggesting that ectopically expressed hTERT associates stably with other factors that are required for telomerase activity. We note that the peak of FlaghTERT coincided with the NVL2 peak in fractions 5–9, further supporting the existence of two complexes of active telomerase.
Recently, the AAA-ATPases pontin and reptin were shown to be essential for telomerase holoenzyme assembly through interactions with both hTERT and dyskerin [22]. We compared the sedimentation profile of NVL2 with the sedimentation profiles of pontin and reptin. Both pontin and reptin sedimented in fractions 5–11 and cosedimented with NVL2 with the peak in fraction number 7 (Fig. 2B and C). These results suggest that NVL2, like pontin and reptin, is not required for telomerase catalytic activity but may serve to assemble a telomerase complex (see the Discussion). 3.3. Knockdown of endogenous NVL2 decreases hTERT level and telomerase activity To examine the role of NVL2 in a more physiological setting, the expression of endogenous NVL2 was depleted using two different small interfering RNA duplexes (siRNA). NVL2 knockdown in HeLa cells led to a clear reduction in hTERT levels (Fig. 3A). We next determined the effect of NVL2 knockdown on telomerase activity. NVL2 knockdown also significantly reduced telomerase activity as measured by the TRAP assay (Fig. 3B). These results indicate that NVL2 is essential for hTERT stability as well as telomerase activity. However, it is not clear whether a decrease in telomerase activity by NVL2 was due to the direct effect of telomerase enzyme or to a NVL2-related decrease in the expression of hTERT or hTERC genes. To address this issue, the impact of NVL2 on gene expression of hTERT and hTERC was evaluated by semiquantitative RT-PCR analysis. No significant difference was observed in steady-state levels of hTERT mRNA and hTERC transcripts in cells expressing NVL2 siRNA and control siRNA (Fig. 3C). In contrast to the effect of NVL2 knockdown, telomerase activity was not altered by overexpression of NVL2 (Fig. 3D), suggesting that the endogenous level of NVL2 is sufficient for telomerase assembly.
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Fig. 3. NVL2 knockdown decreases the hTERT levels and telomerase activity. (A) HeLa cells were co-transfected with hTERT-HA and either control (siControl) or NVL2 siRNA (siNVL2-1 or siNVL2-2). The protein levels of endogenous NVL2 and hTERT-HA were measured by immunoblotting with anti-NVL2 and anti-HA antibodies. The asterisk marks the position of nonspecific proteins. (B) HeLa cells were transfected with control or NVL2 siRNA, and different amounts of lysates were analyzed for telomerase activity by the TRAP assay. To test RNA-dependent extension, RNase A (0.25 mg/ml) was added to the extracts before the primer extension reaction. ITAS represents the internal telomerase assay standard. (C) Representative results of RT-PCR analysis for the expression of hTERT and hTR in HeLa cells transfected with control or NVL2 siRNA. RT-PCR products from each sample were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal. (D) HeLa cells were transfected with NVL2-Flag (or the empty vector) and analyzed for telomerase activity.
3.4. ATP-binding activity of NVL2 is required for hTERT binding and telomerase holoenzyme assembly NVL2 has two ATPase domains, each of which contains the conserved lysine residues, required for ATP-binding activity [17]. The K311M and K628M mutations abolish ATP binding activity in the first and second domains, respectively (Fig. 4A) [19]. To determine the effect of the NVL2 mutations on their interaction with hTERT, HeLa cells were co-transfected with hTERT-HA and either wild-type or mutant NVL2-Flag. Co-immunoprecipitation
experiments revealed that both the K311M and K311,628M mutations severely impaired the interaction between hTERT and NVL2, whereas the wild-type and K628M mutant of NVL2 demonstrated a strong interaction with hTERT (Fig. 4B). These results suggest that ATP-binding activity at the first domain, but not the second domain, is required for the interaction of NVL2 with hTERT. To further investigate the role of ATP-binding at the first domain, the effects of the NVL2 mutations on telomerase assembly were examined. HeLa cells transfected with wild-type or mutant NVL2-Flag were subjected to immunoprecipitation with anti-Flag
Fig. 4. ATP-binding activity is required for telomerase holoenzyme assembly. (A) Schematic representation of NVL2 mutations. UD, NVL2 unique domain; AAA, AAA domain. (B) HeLa cells were co-transfected with hTERT-HA and wild-type or mutant NVL2-Flag and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblotting with anti-HA antibody. (C) HeLa cells were transfected with wild-type or mutant NVL2-Flag and subjected to immunoprecipitation with anti-Flag antibody, followed by analysis for telomerase activity. ITAS represents the internal telomerase assay standard.
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antibody and analyzed for telomerase activity. Telomerase activity was precipitated by anti-Flag antibody in cells expressing wildtype NVL2 and K628M (Fig. 4C). In contrast, the amounts of telomerase activity recovered from cells expressing K311M and K311,628M were significantly reduced. These results suggest that ATP-binding activity at the first domain is essential for assembly of catalytically competent telomerase. 4. Discussion In this study, we describe the characterization of NVL2 as a new protein interacting with hTERT. The following results support that NVL2 is a telomerase component essential for holoenzyme assembly. First, hTERT interacts with NVL2 in the nucleolus but has reduced binding affinity to NVL1 that is nucleoplasmic, suggesting a physiological implication of the interaction of NVL2 with hTERT in the nucleolus. Second, immunofluorescence experiments demonstrate that NVL2 is a nucleolar protein that co-localizes with hTERT in mammalian cells. Third, telomerase activity is immunoprecipitated by NVL2. The amount of telomerase activity was substantially reduced in NVL1 immunoprecipitates, which is consistent with the finding that NVL2 has higher affinity for hTERT binding than NVL1. Fourth, knockdown of endogenous NVL2 reduces hTERT level as well as telomerase activity. Finally, ATP-binding activity of NVL2 is required for hTERT binding and telomerase holoenzyme assembly. Taken together, these results provide an insight into the new cellular function of NVL2 in addition to its role in ribosomal biosynthesis. This novel function could be relevant to its role in cancer progression. Given the complexity of many other RNP enzymes, it is likely that telomerase requires multiple associated proteins for proper assembly and regulation of its enzymatic activity and relies on a step-wise cellular RNP biogenesis pathway to provide assembly specificity [10]. A glycerol gradient sedimentation profile of catalytically competent telomerase revealed that human telomerase exists in at least two different complexes. The NVL2-free telomerase complex has high telomerase catalytic activity, whereas the NVL2-containing complex exhibits lower enzymatic activity (Fig 2B). These results suggest that NVL2 is not required for telomerase catalytic activity but may serve to assemble a telomerase complex containing hTERT. The hTERT-NVL2 complex may represent a pretelomerase complex which requires additional factors for converting to a mature telomerase complex. After assembly, NVL2 should be dissociated from a mature telomerase complex to confer high enzymatic activity. Recently, the related ATPase pontin and reptin were identified as telomerase components through affinity purification with hTERT [22]. Depletion of pontin and reptin markedly impairs telomerase RNP accumulation. Furthermore, although pontin and reptin associate with a significant population of hTERT molecules, they do not yield high-level telomerase activity. Thus, NVL2 has common features with pontin and reptin for regulation of telomerase assembly and enzymatic activity. They may serve to facilitate the assembly of hTERT with a telomerase RNP or remodeling a telomerase complex to mature. Telomerase Cajal body protein 1 (TCAB1) was identified as a telomerase holoenzyme subunit, which is notably enriched in Cajal bodies, nuclear sites of telomerase RNP processing [23]. Depletion of TCAB1 prevents hTERC from associating with Cajal bodies and inhibits the ability of telomerase to elongate telomeric repeats. However, depletion of TCAB1 does not reduce telomerase activity, indicating that an active telomerase complex can form in different nuclear compartment [24]. Indeed, nucleolin has been identified to interact with hTERT, and this binding would be critical for the nucleolar localization of hTERT [25]. We showed that NVL2 associates with active telomerase enzyme through an interaction with hTERT in the nucleolus, and that this binding is essential for
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telomerase assembly. It is very likely that the telomerase holoenzyme is initially assembled in the nucleolus, which in turn translocates into Cajal bodies for elongating telomeres. Telomerase lengthens telomeric repeats preferentially during S phase of the cell cycle [26]. Thus, it will be of interest to determine whether NVL2 contributes to cell cycle control of telomerase assembly. Although many important questions about the physiological role of NVL2 in the assembly of active telomerase remain to be seen, our results suggest that NVL2 represents a new pathway for regulating the telomerase assembly as well as its enzymatic activity and provides an alternative approach for inhibiting telomerase activity in cancer. Acknowledgments This work was supported in part by World Class University Fund (R31-2009-000-10086-0) from the Korean Ministry of Education, Science, and Technology and by Korea Research Foundation Grants (KRF-M1075604000107N560400110). References [1] A. Smogorzewska, T. de Lange, Regulation of telomerase by telomeric proteins, Annu. Rev. Biochem. 73 (2004) 177–208. [2] J.D. Griffith, L. Comeau, S. Rosenfield, R.M. Stansel, A. Bianchi, H. Moss, T. de Lange, Mammalian telomeres end in a large duplex loop, Cell 97 (1999) 503– 514. [3] T. de Lange, Shelterin: the protein complex that shapes and safeguards human telomeres, Genes Dev. 19 (2005) 2100–2110. [4] D. Liu, M.S. O’Connor, J. Qin, Z. Songyang, Telosome, a mammalian telomereassociated complex formed by multiple telomeric proteins, J. Biol. Chem. 279 (2004) 51338–51342. [5] J. Lingner, J.P. Cooper, T.R. Cech, Telomerase and DNA end replication: no longer a lagging strand problem?, Science 269 (1995) 1533–1534 [6] M.A. Dunham, A.A. Neumann, C.L. Fasching, R.R. Reddel, Telomere maintenance by recombination in human cells, Nat. Genet. 26 (2000) 447–450. [7] C. Autexier, N.F. Lue, The structure and function of telomerase reverse transcriptase, Annu. Rev. Biochem. 75 (2006) 493–517. [8] A. Bianchi, D. Shore, How telomerase reaches its end: mechanism of telomerase regulation by the telomeric complex, Mol. Cell 31 (2008) 153–165. [9] N.W. Kim, M.A. Piatyszek, K.R. Prowse, C.B. Harley, M.D. West, P.L. Ho, G.M. Coviello, W.E. Wright, S.L. Weinrich, J.W. Shay, Specific association of human telomerase activity with immortal cells and cancer, Science 266 (1994) 2011– 2015. [10] K. Collins, The biogenesis and regulation of telomerase holoenzymes, Nat. Rev. Mol. Cell. Biol. 7 (2006) 484–494. [11] S.E. Holt, D.L. Aisner, J. Baur, V.M. Tesmer, M. Dy, M. Ouellette, J.B. Trager, G.B. Morin, D.O. Toft, J.W. Shay, W.E. Wright, M.A. White, Functional requirement of p23 and Hsp90 in telomerase complexes, Genes Dev. 13 (1999) 817–826. [12] J.H. Lee, P. Khadka, S.H. Baek, I.K. Chung, CHIP promotes human telomerase reverse transcriptase degradation and negatively regulates telomerase activity, J. Biol. Chem. 285 (2010) 42033–42045. [13] H. Seimiya, H. Sawada, Y. Muramatsu, M. Shimizu, K. Ohko, K. Yamane, T. Tsuruo, Involvement of 14-3-3 proteins in nuclear localization of telomerase, EMBO J. 19 (2000) 2652–2661. [14] M. Akiyama, T. Hideshima, T. Hayashi, Y.T. Tai, C.S. Mitsiades, N. Mitsiades, D. Chauhan, P. Richardson, N.C. Munshi, K.C. Anderson, Nuclear factor-B p65 mediates tumor necrosis factor alpha-induced nuclear translocation of telomerase reverse transcriptase protein, Cancer Res. 63 (2003) 18–21. [15] X.Z. Zhou, K.P. Lu, The Pin2/TRF1-interacting protein PinX1 is a potent telomerase inhibitor, Cell 107 (2001) 347–359. [16] G.E. Lee, E.Y. Yu, C.H. Cho, J. Lee, M.T. Muller, I.K. Chung, DNA-protein kinase catalytic subunit-interacting protein KIP binds telomerase by interacting with human telomerase reverse transcriptase, J. Biol. Chem. 279 (2004) 34750– 34755. [17] E.L. Germain-Lee, C. Obie, D. Valle, NVL: a new member of the AAA family of ATPases localized to the nucleus, Genomics 44 (1997) 22–34. [18] M. Nagahama, Y. Hara, A. Seki, T. Yamazoe, Y. Kawate, T. Shinohara, K. Hatsuzawa, K. Tani, M. Tagaya, NVL2 is a nucleolar AAA-ATPase that interacts with ribosomal protein L5 through its nucleolar localization sequence, Mol. Biol. Cell 15 (2004) 5712–5723. [19] M. Nagahama, T. Yamazoe, Y. Hara, K. Tani, A. Tsuji, M. Tagaya, The AAAATPase NVL2 is a component of pre-ribosomal particles that interacts with the DExD/H-box RNA helicase DOB1, Biochem. Biophys. Res. Commun. 346 (2006) 1075–1082. [20] Y. Fujiwara, K. Fujiwara, N. Goda, N. Iwaya, T. Tenno, M. Shirakawa, H. Hiroaki, Structure and function of the N-terminal nucleolin binding domain of nuclear valosin-containing protein-like 2 (NVL2) harboring a nucleolar localization signal, J. Biol. Chem. 286 (2011) 21732–21741.
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The AAA-ATPase NVL2 is a telomerase component essential for holoenzyme assembly Joonyoung Her, In Kwon Chung ⇑ Departments of Biology and Integrated Omics for Biomedical Science, Yonsei University, Seoul 120-749, South Korea
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Article history: Received 19 December 2011 Available online 27 December 2011 Keywords: Telomerase hTERT NVL2 AAA-ATPase Nucleolus
a b s t r a c t Continued cell proliferation requires telomerase to maintain functional telomeres that are essential for chromosome integrity. Although the core enzyme includes a telomerase reverse transcriptase (TERT) and a telomerase RNA component (TERC), a number of auxiliary proteins have been identified to regulate telomerase assembly, localization, and enzymatic activity. Here we describe the characterization of the AAA-ATPase NVL2 as a novel hTERT-interacting protein. NVL2 interacts and co-localizes with hTERT in the nucleolus. NLV2 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT. Depletion of endogenous NVL2 by small interfering RNA led to a decrease in hTERT without affecting the steady-state levels of hTERT mRNA, thereby reducing telomerase activity, suggesting that NVL2 is an essential component of the telomerase holoenzyme. We also found that ATP-binding activity of NVL2 is required for hTERT binding as well as telomerase assembly. Our findings suggest that NVL2, in addition to its role in ribosome biosynthesis, is essential for telomerase biogenesis and provides an alternative approach for inhibiting telomerase activity in cancer. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Telomeres are essential and functional components located at the physical ends of eukaryotic chromosomes and are responsible for maintaining chromosome stability [1]. In most eukaryotic organisms, telomeres consist of long tracts of duplex telomere repeats (TTAGGG in vertebrates) with 30 single-stranded G overhang [2] and proteins that associate directly and indirectly with telomeric DNA sequences [3,4]. In the absence of functional telomere maintenance pathways, dividing cells show a progressive loss of telomeric DNA during each round of cell division [5]. Although recombination-based telomere elongation has been demonstrated for replenishing telomeric DNA [6], the major mechanism to compensate for telomere loss involves ongoing elongation of telomeric repeats by telomerase [7,8]. Telomerase activity is observed in most tumor cells but undetectable in normal somatic cells [9], suggesting an attractive molecular target for anti-cancer therapy. Although the core enzyme includes a telomerase reverse transcriptase (TERT) and a telomerase RNA component (TERC) [10], a number of auxiliary proteins have been identified to regulate telomerase assembly [11,12], localization [13,14], and enzymatic activity [15,16].
⇑ Corresponding author. Address: Department of Integrated Omics for Biomedical Science, Yonsei University, 134 Shinchon-dong, Seoul 120-749, South Korea. Fax: +822 364 8660. E-mail address: [email protected] (I.K. Chung). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.12.101
In a search for proteins capable of interacting with human telomerase, we identified nuclear valosin-containing protein-like (NVL) as an hTERT-interacting partner in a yeast two-hybrid screen. NVL was first identified as a member of the chaperone-like AAA-ATPase (for ATPase associated with various cellular activities) family with two conserved ATP-binding domains, which displays a high level of amino acid similarity with a cytosolic chaperone, VCP/ p97 [17]. NVL exists as two alternatively spliced forms, NVL1 (a short form) and NVL2 (a long form), which are produced from different methionines as the translation initiation sites. Whereas NVL1 is present in the nucleoplasm, NVL2 is mainly localized in the nucleolus where it is involved in the biogenesis of the 60S ribosomal subunit [18]. In this process, NVL2 has been shown to associate with ribosomal protein L5 [18] and RNA helicase DOB1 [19]. More recently, NVL2 was reported to interact with nucleolin through its N-terminal unique domain and facilitate the recycling of nucleolin using its ATPase activity for efficient ribosome biogenesis [20]. Here we report the characterization of NVL2 as an hTERT-interacting protein. NVL2 physically interacts and co-localizes with hTERT in the nucleolus. NVL2 also associates with catalytically competent telomerase through an interaction with hTERT. Whereas overexpression of NVL2 has no effect on telomerase activity, depletion of endogenous NVL2 in telomerase-positive cells led to a decrease in hTERT without affecting the steady-state levels of hTERT mRNA, thereby reducing telomerase activity. Overall, our data suggest that NVL2, in addition to its participation in ribosome
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biogenesis, is required for telomerase assembly and has a unique and independent role in the regulation of telomerase activity. Hence, NVL2 represents an alternative pathway for modulating telomerase activity in cancer.
35,000 rpm for 15 h at 4 °C in a linear 10–30% (v/v) glycerol gradient in a Beckman SW41Ti rotor. Fractions of 0.5 ml each were collected from the bottom of the gradient, and 15 ll of each fraction was analyzed for immunoblots.
2. Materials and methods
2.6. RNA Interference
2.1. Cell culture and plasmids
The siRNA target sequences specific for NVL were 50 -GACUCGUUAGACCCUGCUU-30 for siNVL-1 and 50 -GGCAAGACAGAAGAG UGGAAAUGAA-30 for siNVL-2. The siRNA duplexes were transfected into HeLa cells using RNAiMax transfection reagent (Invitrogen). A scramble sequence (50 -AATCGCATAGCGTATGCCGTT-30 ) was used as a control that did not correspond to any known gene.
HeLa and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 lg/ml streptomycin in 5% CO2 at 37 °C. The 3 FLAG-hTERT expression vector was constructed by inserting the full-length hTERT cDNA into pCMV7.1 3 FLAG vector (Sigma), and the hTERT-HA expression vector has been described previously [13]. Mammalian expression plasmids for FLAG-tagged wild-type NVL2 and mutants (K311M and K628M) were kind gifts from Dr. Mitsuo Tagaya (Tokyo University of Pharmacy and Life Science, Japan) [19]. The K311,628M double point mutant was generated by site-direct mutagenesis. The NVL1-V5 and NVL2-V5 expression vectors were constructed by inserting the PCR fragments into pcDNA3.1/V5-His (Invitrogen). The expression vectors for GSTNVL2 were constructed by cloning the full-length and truncated proteins from the NVL2 cDNA into pGEX-4T-1 (GE Healthcare). 2.2. GST pull-down, immunoprecipitation, and immunoblot analyses GST pull-down and immunoprecipitation were performed as previously described [16]. Immunoprecipitation and immunoblot analyses were performed using anti-NVL (Abnova), anti-FLAG (Sigma), anti-HA (Santa Cruz Biotechnology), anti-V5 (Invitrogen), anti-Reptin (Santa Cruz Biotechnology), anti-pontin (Santa Cruz Biotechnology), anti-tubulin (Santa Cruz Biotechnology), and antiactin (Sigma) antibodies as specified. Immunoblotting experiments were repeated at least three times and a representative blot is shown. 2.3. Telomerase assay The telomeric repeat amplification protocol (TRAP) was used as previously described [21]. Briefly, cell extracts (200 ng of protein) were added to the telomerase extension reactions and incubated for 20 min at 37 °C, and PCR was performed using the HTS primer and HACX primer for 30 cycles. As an internal telomerase assay standard, NT and TSNT primers were added to the PCR mixture. 2.4. Immunofluorescence microscopy Cells were grown on glass coverslips fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min and permeabilized in 0.2% Triton X-100 in PBS for 20 min. Cells were then blocked in 5% bovine serum albumin and incubated with goat anti-hTERT (Santa Cruz Biotechnology), mouse anti-NVL (Abnova) or rabbit anti-nucleolin (Santa Cruz Biotechnology) antibodies overnight at 4 °C. After washing with PBS, cells were incubated with Alexa Fluor 488-labeled rabbit anti-goat or goat anti-rabbit immuoglobulin and Alexa Fluor 546-labeled goat anti-mouse immunoglobulin (Molecular Probes). DNA was stained with 4,6diamino-2-phenylindole (Vectashield, Vector Laboratories) for 1 h at room temperature. Immunofluorescence images were captured using a confocal laser-scanning microscope (Carl Zeiss). 2.5. Glycerol gradient sedimentation Glycerol gradient sedimentation experiments were performed as previously described [22]. Briefly, extracts were centrifuged at
3. Results 3.1. Identification of NVL2 as an hTERT-interacting partner To identify hTERT-interacting factors, we screened a HeLa cell cDNA library using the yeast two-hybrid system [16]. With the carboxy-terminal domain of hTERT (amino acids 946-1132) as bait, a clone containing the full-length NVL2 cDNA was obtained and sequenced. NVL was first identified as a gene product that displays a high level of amino acid similarity with an AAA-ATPase, VCP/p97 [17] and has two alternatively spliced isoforms. Because NLV2 is exclusively localized in the nucleolus where the telomerase ribonucleoprotein (RNP) complex is assembled, we are particularly interested in identification of a nucleolar protein that can associate with hTERT. To confirm the direct interaction between hTERT and NVL2, we performed GST pulldown experiments. GST-NLV2, but not GSThRAP1 or the control GST, precipitated hTERT-HA expressed in HeLa cells, indicating that NVL2 interacts with hTERT in vitro (Fig. 1A). To determine whether hTERT and NVL2 associate in vivo, HeLa cells were co-transfected with hTERT-HA and NVL2Flag expression vectors and subjected to immunoprecipitation. hTERT-HA was detected in anti-Flag immunoprecipitates when NVL2-Flag was expressed (Fig. 1B). Likewise, NVL2-Flag was recovered in anti-HA immunoprecipitates when hTERT-HA was expressed. Endogenous NVL2 was also immunoprecipitated by hTERT-HA (Fig. 1C), indicating that NVL2 interacts with hTERT in mammalian cells. To map the region in NVL2 that is important for hTERT binding, a series of NVL2 fragments were fused to GST and used in the in vitro binding assay (Fig. 1D). Full-length NVL1 and NVL2 were found to associate with hTERT while other truncated forms failed to interact with hTERT (Fig. 1E). NVL2 has much higher affinity for hTERT binding than its shorter form, NVL1. We next determined the subcellular localization of NVL2 and hTERT by immunofluorescence staining. Endogenous NVL2 signals were almost exclusively detected in the nucleolus, as demonstrated by the finding that nucleolin clearly co-localized with NVL2 (Fig. 1F). Although some found in the nucleoplasm, hTERT was predominantly co-localized with NVL2 in the nucleolus (Fig. 1G). Given that telomerase ribonucleoprotein (RNP) is assembled in the nucleolus before translocating into the cajal body [23,24], the observations that NVL2 interacts and co-localizes with hTERT in the nucleolus suggest that NVL2 may participate in telomerase biogenesis. 3.2. NVL2 associates with catalytically competent telomerase Because NVL2 interacts with hTERT, we examined whether NVL2 associates with catalytically competent telomerase. HeLa cells transfected with NVL1-V5 or NVL2-V5 (or the empty vector) were subjected to immunoprecipitation with anti-V5 antibody and analyzed for telomerase activity by the telomerase repeat
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Fig. 1. NVL2 interacts and co-localizes with hTERT. (A) GST, GST-NVL2, or GST-hRAP1 were immobilized on glutathione-Sepharose and incubated with ectopically expressed hTERT-HA. Bound proteins were detected by immunoblotting with anti-HA antibody. (B) HeLa cells were co-transfected with hTERT-HA and NVL2-Flag and subjected to immunoprecipitation with anti-Flag or anti-HA antibodies, followed by immunoblotting as indicated. The asterisk marks the position of nonspecific proteins. (C) HeLa cells were transfected with hTERT-HA and subjected to immunoprecipitation with anti-HA antibody, followed by immunoblotting with anti-NVL2 antibody. (D) Schematic representation of NVL2 truncations fused to GST. The numbering of amino acids starts from the initiation methionine of NVL2. UD, NVL2 unique domain; AAA, AAA domain. (E) GST or the various truncated GST-NVL2 fusion proteins were affinity-purified and incubated with ectopically expressed hTERT-HA. The purified GST fusion proteins were visualized by Coomassie staining and indicated with arrowheads. Molecular mass markers are shown in kilodaltons. (F) HeLa cells were analyzed by immunofluorescence for co-localization of NVL2 with nucleolin. Immunofluorescence was used to detect endogenous NVL2 (red) and nucleolin (green). DNA was stained with 4,6-diamidino-2phenylindole (DAPI) (blue). (G) Immunofluorescence was used to detect endogenous NVL2 (red) and hTERT (green). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. NVL2 associates with catalytically competent telomerase. (A) HeLa cells transfected with NVL1-V5 or NVL2-V5 (or the empty vector) were subjected to immunoprecipitation with anti-V5 antibody and analyzed for telomerase activity by the TRAP assay. ITAS represents the internal telomerase assay standard. (B) Extracts from HEK293 cells were fractionated through 10–30% (v/v) glycerol gradients. Fractions were subjected to Western blotting to visualize NVL2, reptin, and pontin and analyzed for telomerase activity. (C) Extracts from HEK293 cells expressing Flag-hTERT were subjected to sedimentation analysis.
amplification protocol (TRAP) assay. Telomerase activity was detected in anti-V5 immunoprecipitates in cells expressing NVL1 and NVL2 but not in the control cells (Fig. 2A). The amount of telomerase activity recovered from NVL2-expressing cells was significantly higher than that recovered from NVL1-expressing cells even though the higher level of NVL1-V5 was expressed compared to NVL2-V5. This could be due to higher affinity of NVL2 for hTERT binding as shown in Fig. 1E. These results indicate that NVL2 associates with catalytically competent telomerase through an interaction with hTERT, and that this association could be specific. To further confirm that NVL2 associates with telomerase complex, extracts from HEK293 cells were subjected to sedimentation analysis on a linear 10–30% (v/v) glycerol gradient. Western blot analysis revealed that endogenous NVL2 was fractionated as a single peak spanning fractions 5–9 (Fig. 2B). Endogenous telomerase activity sedimented in fractions 1–9 with the peak centered on fraction number 5. Whereas telomerase activity cosedimented with NVL2 in fractions 5–9, NVL2 signal was not detected in fractions 1–3 (Fig. 2B). Because telomerase activity was immunoprecipitated by NVL2, these results suggest that catalytically competent telomerase exists in at least two different complexes; one complex contains NVL2 (fractions 5–7), and the other is free from NVL2 (fractions 1–3). We next wished to determine the sedimentation behavior of hTERT. However, we could not detect endogenous hTERT possibly due to its dilution in glycerol gradient fractions. Thus, HEK293 cells were transfected with Flag-hTERT and subjected to sedimentation analysis under identical conditions (Fig. 2C). Flag-hTERT sedimented in fractions 1–11 with the peak in fraction number 5, suggesting that ectopically expressed hTERT associates stably with other factors that are required for telomerase activity. We note that the peak of FlaghTERT coincided with the NVL2 peak in fractions 5–9, further supporting the existence of two complexes of active telomerase.
Recently, the AAA-ATPases pontin and reptin were shown to be essential for telomerase holoenzyme assembly through interactions with both hTERT and dyskerin [22]. We compared the sedimentation profile of NVL2 with the sedimentation profiles of pontin and reptin. Both pontin and reptin sedimented in fractions 5–11 and cosedimented with NVL2 with the peak in fraction number 7 (Fig. 2B and C). These results suggest that NVL2, like pontin and reptin, is not required for telomerase catalytic activity but may serve to assemble a telomerase complex (see the Discussion). 3.3. Knockdown of endogenous NVL2 decreases hTERT level and telomerase activity To examine the role of NVL2 in a more physiological setting, the expression of endogenous NVL2 was depleted using two different small interfering RNA duplexes (siRNA). NVL2 knockdown in HeLa cells led to a clear reduction in hTERT levels (Fig. 3A). We next determined the effect of NVL2 knockdown on telomerase activity. NVL2 knockdown also significantly reduced telomerase activity as measured by the TRAP assay (Fig. 3B). These results indicate that NVL2 is essential for hTERT stability as well as telomerase activity. However, it is not clear whether a decrease in telomerase activity by NVL2 was due to the direct effect of telomerase enzyme or to a NVL2-related decrease in the expression of hTERT or hTERC genes. To address this issue, the impact of NVL2 on gene expression of hTERT and hTERC was evaluated by semiquantitative RT-PCR analysis. No significant difference was observed in steady-state levels of hTERT mRNA and hTERC transcripts in cells expressing NVL2 siRNA and control siRNA (Fig. 3C). In contrast to the effect of NVL2 knockdown, telomerase activity was not altered by overexpression of NVL2 (Fig. 3D), suggesting that the endogenous level of NVL2 is sufficient for telomerase assembly.
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Fig. 3. NVL2 knockdown decreases the hTERT levels and telomerase activity. (A) HeLa cells were co-transfected with hTERT-HA and either control (siControl) or NVL2 siRNA (siNVL2-1 or siNVL2-2). The protein levels of endogenous NVL2 and hTERT-HA were measured by immunoblotting with anti-NVL2 and anti-HA antibodies. The asterisk marks the position of nonspecific proteins. (B) HeLa cells were transfected with control or NVL2 siRNA, and different amounts of lysates were analyzed for telomerase activity by the TRAP assay. To test RNA-dependent extension, RNase A (0.25 mg/ml) was added to the extracts before the primer extension reaction. ITAS represents the internal telomerase assay standard. (C) Representative results of RT-PCR analysis for the expression of hTERT and hTR in HeLa cells transfected with control or NVL2 siRNA. RT-PCR products from each sample were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal. (D) HeLa cells were transfected with NVL2-Flag (or the empty vector) and analyzed for telomerase activity.
3.4. ATP-binding activity of NVL2 is required for hTERT binding and telomerase holoenzyme assembly NVL2 has two ATPase domains, each of which contains the conserved lysine residues, required for ATP-binding activity [17]. The K311M and K628M mutations abolish ATP binding activity in the first and second domains, respectively (Fig. 4A) [19]. To determine the effect of the NVL2 mutations on their interaction with hTERT, HeLa cells were co-transfected with hTERT-HA and either wild-type or mutant NVL2-Flag. Co-immunoprecipitation
experiments revealed that both the K311M and K311,628M mutations severely impaired the interaction between hTERT and NVL2, whereas the wild-type and K628M mutant of NVL2 demonstrated a strong interaction with hTERT (Fig. 4B). These results suggest that ATP-binding activity at the first domain, but not the second domain, is required for the interaction of NVL2 with hTERT. To further investigate the role of ATP-binding at the first domain, the effects of the NVL2 mutations on telomerase assembly were examined. HeLa cells transfected with wild-type or mutant NVL2-Flag were subjected to immunoprecipitation with anti-Flag
Fig. 4. ATP-binding activity is required for telomerase holoenzyme assembly. (A) Schematic representation of NVL2 mutations. UD, NVL2 unique domain; AAA, AAA domain. (B) HeLa cells were co-transfected with hTERT-HA and wild-type or mutant NVL2-Flag and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblotting with anti-HA antibody. (C) HeLa cells were transfected with wild-type or mutant NVL2-Flag and subjected to immunoprecipitation with anti-Flag antibody, followed by analysis for telomerase activity. ITAS represents the internal telomerase assay standard.
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antibody and analyzed for telomerase activity. Telomerase activity was precipitated by anti-Flag antibody in cells expressing wildtype NVL2 and K628M (Fig. 4C). In contrast, the amounts of telomerase activity recovered from cells expressing K311M and K311,628M were significantly reduced. These results suggest that ATP-binding activity at the first domain is essential for assembly of catalytically competent telomerase. 4. Discussion In this study, we describe the characterization of NVL2 as a new protein interacting with hTERT. The following results support that NVL2 is a telomerase component essential for holoenzyme assembly. First, hTERT interacts with NVL2 in the nucleolus but has reduced binding affinity to NVL1 that is nucleoplasmic, suggesting a physiological implication of the interaction of NVL2 with hTERT in the nucleolus. Second, immunofluorescence experiments demonstrate that NVL2 is a nucleolar protein that co-localizes with hTERT in mammalian cells. Third, telomerase activity is immunoprecipitated by NVL2. The amount of telomerase activity was substantially reduced in NVL1 immunoprecipitates, which is consistent with the finding that NVL2 has higher affinity for hTERT binding than NVL1. Fourth, knockdown of endogenous NVL2 reduces hTERT level as well as telomerase activity. Finally, ATP-binding activity of NVL2 is required for hTERT binding and telomerase holoenzyme assembly. Taken together, these results provide an insight into the new cellular function of NVL2 in addition to its role in ribosomal biosynthesis. This novel function could be relevant to its role in cancer progression. Given the complexity of many other RNP enzymes, it is likely that telomerase requires multiple associated proteins for proper assembly and regulation of its enzymatic activity and relies on a step-wise cellular RNP biogenesis pathway to provide assembly specificity [10]. A glycerol gradient sedimentation profile of catalytically competent telomerase revealed that human telomerase exists in at least two different complexes. The NVL2-free telomerase complex has high telomerase catalytic activity, whereas the NVL2-containing complex exhibits lower enzymatic activity (Fig 2B). These results suggest that NVL2 is not required for telomerase catalytic activity but may serve to assemble a telomerase complex containing hTERT. The hTERT-NVL2 complex may represent a pretelomerase complex which requires additional factors for converting to a mature telomerase complex. After assembly, NVL2 should be dissociated from a mature telomerase complex to confer high enzymatic activity. Recently, the related ATPase pontin and reptin were identified as telomerase components through affinity purification with hTERT [22]. Depletion of pontin and reptin markedly impairs telomerase RNP accumulation. Furthermore, although pontin and reptin associate with a significant population of hTERT molecules, they do not yield high-level telomerase activity. Thus, NVL2 has common features with pontin and reptin for regulation of telomerase assembly and enzymatic activity. They may serve to facilitate the assembly of hTERT with a telomerase RNP or remodeling a telomerase complex to mature. Telomerase Cajal body protein 1 (TCAB1) was identified as a telomerase holoenzyme subunit, which is notably enriched in Cajal bodies, nuclear sites of telomerase RNP processing [23]. Depletion of TCAB1 prevents hTERC from associating with Cajal bodies and inhibits the ability of telomerase to elongate telomeric repeats. However, depletion of TCAB1 does not reduce telomerase activity, indicating that an active telomerase complex can form in different nuclear compartment [24]. Indeed, nucleolin has been identified to interact with hTERT, and this binding would be critical for the nucleolar localization of hTERT [25]. We showed that NVL2 associates with active telomerase enzyme through an interaction with hTERT in the nucleolus, and that this binding is essential for
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telomerase assembly. It is very likely that the telomerase holoenzyme is initially assembled in the nucleolus, which in turn translocates into Cajal bodies for elongating telomeres. Telomerase lengthens telomeric repeats preferentially during S phase of the cell cycle [26]. Thus, it will be of interest to determine whether NVL2 contributes to cell cycle control of telomerase assembly. Although many important questions about the physiological role of NVL2 in the assembly of active telomerase remain to be seen, our results suggest that NVL2 represents a new pathway for regulating the telomerase assembly as well as its enzymatic activity and provides an alternative approach for inhibiting telomerase activity in cancer. Acknowledgments This work was supported in part by World Class University Fund (R31-2009-000-10086-0) from the Korean Ministry of Education, Science, and Technology and by Korea Research Foundation Grants (KRF-M1075604000107N560400110). References [1] A. Smogorzewska, T. de Lange, Regulation of telomerase by telomeric proteins, Annu. Rev. Biochem. 73 (2004) 177–208. [2] J.D. Griffith, L. Comeau, S. Rosenfield, R.M. Stansel, A. Bianchi, H. Moss, T. de Lange, Mammalian telomeres end in a large duplex loop, Cell 97 (1999) 503– 514. [3] T. de Lange, Shelterin: the protein complex that shapes and safeguards human telomeres, Genes Dev. 19 (2005) 2100–2110. [4] D. Liu, M.S. O’Connor, J. Qin, Z. Songyang, Telosome, a mammalian telomereassociated complex formed by multiple telomeric proteins, J. Biol. Chem. 279 (2004) 51338–51342. [5] J. Lingner, J.P. Cooper, T.R. Cech, Telomerase and DNA end replication: no longer a lagging strand problem?, Science 269 (1995) 1533–1534 [6] M.A. Dunham, A.A. Neumann, C.L. Fasching, R.R. Reddel, Telomere maintenance by recombination in human cells, Nat. Genet. 26 (2000) 447–450. [7] C. Autexier, N.F. Lue, The structure and function of telomerase reverse transcriptase, Annu. Rev. Biochem. 75 (2006) 493–517. [8] A. Bianchi, D. Shore, How telomerase reaches its end: mechanism of telomerase regulation by the telomeric complex, Mol. Cell 31 (2008) 153–165. [9] N.W. Kim, M.A. Piatyszek, K.R. Prowse, C.B. Harley, M.D. West, P.L. Ho, G.M. Coviello, W.E. Wright, S.L. Weinrich, J.W. Shay, Specific association of human telomerase activity with immortal cells and cancer, Science 266 (1994) 2011– 2015. [10] K. Collins, The biogenesis and regulation of telomerase holoenzymes, Nat. Rev. Mol. Cell. Biol. 7 (2006) 484–494. [11] S.E. Holt, D.L. Aisner, J. Baur, V.M. Tesmer, M. Dy, M. Ouellette, J.B. Trager, G.B. Morin, D.O. Toft, J.W. Shay, W.E. Wright, M.A. White, Functional requirement of p23 and Hsp90 in telomerase complexes, Genes Dev. 13 (1999) 817–826. [12] J.H. Lee, P. Khadka, S.H. Baek, I.K. Chung, CHIP promotes human telomerase reverse transcriptase degradation and negatively regulates telomerase activity, J. Biol. Chem. 285 (2010) 42033–42045. [13] H. Seimiya, H. Sawada, Y. Muramatsu, M. Shimizu, K. Ohko, K. Yamane, T. Tsuruo, Involvement of 14-3-3 proteins in nuclear localization of telomerase, EMBO J. 19 (2000) 2652–2661. [14] M. Akiyama, T. Hideshima, T. Hayashi, Y.T. Tai, C.S. Mitsiades, N. Mitsiades, D. Chauhan, P. Richardson, N.C. Munshi, K.C. Anderson, Nuclear factor-B p65 mediates tumor necrosis factor alpha-induced nuclear translocation of telomerase reverse transcriptase protein, Cancer Res. 63 (2003) 18–21. [15] X.Z. Zhou, K.P. Lu, The Pin2/TRF1-interacting protein PinX1 is a potent telomerase inhibitor, Cell 107 (2001) 347–359. [16] G.E. Lee, E.Y. Yu, C.H. Cho, J. Lee, M.T. Muller, I.K. Chung, DNA-protein kinase catalytic subunit-interacting protein KIP binds telomerase by interacting with human telomerase reverse transcriptase, J. Biol. Chem. 279 (2004) 34750– 34755. [17] E.L. Germain-Lee, C. Obie, D. Valle, NVL: a new member of the AAA family of ATPases localized to the nucleus, Genomics 44 (1997) 22–34. [18] M. Nagahama, Y. Hara, A. Seki, T. Yamazoe, Y. Kawate, T. Shinohara, K. Hatsuzawa, K. Tani, M. Tagaya, NVL2 is a nucleolar AAA-ATPase that interacts with ribosomal protein L5 through its nucleolar localization sequence, Mol. Biol. Cell 15 (2004) 5712–5723. [19] M. Nagahama, T. Yamazoe, Y. Hara, K. Tani, A. Tsuji, M. Tagaya, The AAAATPase NVL2 is a component of pre-ribosomal particles that interacts with the DExD/H-box RNA helicase DOB1, Biochem. Biophys. Res. Commun. 346 (2006) 1075–1082. [20] Y. Fujiwara, K. Fujiwara, N. Goda, N. Iwaya, T. Tenno, M. Shirakawa, H. Hiroaki, Structure and function of the N-terminal nucleolin binding domain of nuclear valosin-containing protein-like 2 (NVL2) harboring a nucleolar localization signal, J. Biol. Chem. 286 (2011) 21732–21741.
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