Nucleolin interacts with telomerase

Abstract

Telomerase is a specialized reverse transcriptase composed of core RNA and protein subunits which plays essential roles in maintaining telomeres in actively dividing cells. Recent work indicates that telomerase shuttles between subcellular compartments during assembly and in response to specific stimuli. In particular, telomerase colocalizes with nucleoli in normal human fibroblasts. Here, we show that nucleolin, a major nucleolar phosphoprotein, interacts with telomerase and alters its subcellular localization. Nucleolin binds the human telomerase reverse transcriptase subunit (hTERT) through interactions with its RNA binding domain 4 and carboxyl-terminal RGG domain, and this binding also involves the telomerase RNA subunit hTERC. The protein-protein interaction between nucleolin and hTERT is critical for the nucleolar localization of hTERT. These findings indicate that interaction of hTERT and nucleolin participates in the dynamic intracellular localization of telomerase complex. Telomerase is a specialized reverse transcriptase composed of core RNA and protein subunits which plays essential roles in maintaining telomeres in actively dividing cells. Recent work indicates that telomerase shuttles between subcellular compartments during assembly and in response to specific stimuli. In particular, telomerase colocalizes with nucleoli in normal human fibroblasts. Here, we show that nucleolin, a major nucleolar phosphoprotein, interacts with telomerase and alters its subcellular localization. Nucleolin binds the human telomerase reverse transcriptase subunit (hTERT) through interactions with its RNA binding domain 4 and carboxyl-terminal RGG domain, and this binding also involves the telomerase RNA subunit hTERC. The protein-protein interaction between nucleolin and hTERT is critical for the nucleolar localization of hTERT. These findings indicate that interaction of hTERT and nucleolin participates in the dynamic intracellular localization of telomerase complex. Telomeres are maintained by the ribonucleoprotein (RNP) 1The abbreviations used are: RNP, ribonucleoprotein; ALT, alternative lengthening of telomere; EGFP, enhanced green fluorescence protein; GST, glutathione S-transferase; hTERC, human telomerase RNA subunit; hTERT, human telomerase reverse transcriptase subunit; PBS, phosphate-buffered saline; RBD, RNA binding domain; RdRP, RNA-dependent RNA polymerase; RGG, RGG rich domain of nucleolin; TRAP, telomere repeat amplification protocol. reverse transcriptase telomerase (1Nakamura T.M. Cech T.R. Cell. 1998; 92: 587-590Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). The enzymatic core of human telomerase RNP is minimally composed of reverse transcriptase catalytic subunit (hTERT) and RNA component (hTERC) (2Weinrich S.L. Pruzan R. Ma L. Ouellette M. Tesmer V.M. Holt S.E. Bodnar A.G. Lichtsteiner S. Kim N.W. Trager J.B. Taylor R.D. Carlos R. Andrews W.H. Wright W.E. Shay J.W. Harley C.B. Morin G.B. Nat. Genet. 1997; 17: 498-502Crossref PubMed Scopus (862) Google Scholar, 3Beattie T.L. Zhou W. Robinson M. Harrington L. Curr. Biol. 1998; 8: 177-180Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 4Feng J. Funk W.D. Wang S.-S. Weinrich S.L. Avilion A.A. Chiu C.-P. Adams R.R. Chang E. Allsopp R.C. Yu J. Le S. West M.D. Harley C.B. Andrews W.H. Greider C.W. Villeponteau B. Science. 1995; 269: 1236-1241Crossref PubMed Scopus (2087) Google Scholar, 5Masutomi K. Kaneko S. Hayashi N. Yamashita T. Shirota Y. Kobayashi K. Murakami S. J. Biol. Chem. 2000; 275: 22568-22573Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar); however, additional factors also regulate telomerase function in vivo (6Counter C.M. Hahn W.C. Wei W. Caddle S.D. Beijersbergen R.L. Lansdorp P.M. Sedivy J.M. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14723-14728Crossref PubMed Scopus (564) Google Scholar, 7Armbruster B.N. Banik S.S. Guo C. Smith A.C. Counter C.M. Mol. Cell. Biol. 2001; 21: 7775-7786Crossref PubMed Scopus (144) Google Scholar, 8Banik S.S. Guo C. Smith A.C. Margolis S.S. Richardson D.A. Tirado C.A. Counter C.M. Mol. Cell. Biol. 2002; 22: 6234-6246Crossref PubMed Scopus (83) Google Scholar). Although expression of hTERT is a rate-limiting step for enzymatic activity of telomerase, additional regulatory steps, such as the assembly of hTERC and hTERT (9Holt S.E. Aisner D.L. Baur J. Tesmer V.M. Dy M. Ouellette M. Toft D.O. Trager J.B. Morin G.B. Wright W.E. Shay J.W. White M.A. Genes Dev. 1999; 13: 817-827Crossref PubMed Scopus (477) Google Scholar, 10Forsythe H.L. Jarvis J.L. Turner J.W. Elmore L.W. Holt S.E. J. Biol. Chem. 2001; 276: 15571-15574Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) and intracellular trafficking of a telomerase complex (11Wong J.M. Kusdra L. Collins K. Nat. Cell Biol. 2002; 4: 731-736Crossref PubMed Scopus (216) Google Scholar, 12Yang Y. Chen Y. Zhang C. Huang H. Weissman S.M. Exp. Cell Res. 2002; 277: 201-209Crossref PubMed Scopus (97) Google Scholar, 13Etheridge K.T. Banik S.S. Armbruster B.N. Zhu Y. Terns R.M. Terns M.P. Counter C.M. J. Biol. Chem. 2002; 277: 24764-24770Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), play critical roles in the assembly and delivery of a biologically active telomerase complex to telomeres. Much of the assembly of ribosomes and possibly other RNPs occurs in nucleoli (14Olson M.O. Dundr M. Szebeni A. Trends Cell Biol. 2000; 10: 189-196Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 15Pederson T. Politz J.C. J. Cell Biol. 2000; 148: 1091-1095Crossref PubMed Scopus (102) Google Scholar). Both hTERT and hTERC localize to nucleoli (11Wong J.M. Kusdra L. Collins K. Nat. Cell Biol. 2002; 4: 731-736Crossref PubMed Scopus (216) Google Scholar, 12Yang Y. Chen Y. Zhang C. Huang H. Weissman S.M. Exp. Cell Res. 2002; 277: 201-209Crossref PubMed Scopus (97) Google Scholar, 13Etheridge K.T. Banik S.S. Armbruster B.N. Zhu Y. Terns R.M. Terns M.P. Counter C.M. J. Biol. Chem. 2002; 277: 24764-24770Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 16Mitchell J.R. Cheng J. Collins K. Mol. Cell. Biol. 1999; 19: 567-576Crossref PubMed Scopus (437) Google Scholar, 17Narayanan A. Lukowiak A. Jady B.E. Dragon F. Kiss T. Terns R.M. Terns M.P. EMBO J. 1999; 18: 5120-5130Crossref PubMed Scopus (109) Google Scholar, 18Lukowiak A.A. Narayanan A. Li Z.H. Terns R.M. Terns M.P. RNA (N. Y.). 2001; 7: 1833-1844PubMed Google Scholar). Nucleolin is a major nucleolar phosphoprotein, and nucleolin-specific antibodies have been used to identify nucleoli (19Tuteja R. Tuteja N. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 407-436Crossref PubMed Scopus (153) Google Scholar, 20Ginisty H. Sicard H. Roger B. Bouvet P. J. Cell Sci. 1999; 112: 761-772Crossref PubMed Google Scholar, 21Srivastava M. Pollard H.B. FASEB J. 1999; 13: 1911-1922Crossref PubMed Scopus (430) Google Scholar). Several studies implicate nucleolin as an RNA chaperone and/or shuttling protein for various host and viral components in nucleoli, the nucleoplasm, cytoplasm, and plasma membrane (19Tuteja R. Tuteja N. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 407-436Crossref PubMed Scopus (153) Google Scholar, 20Ginisty H. Sicard H. Roger B. Bouvet P. J. Cell Sci. 1999; 112: 761-772Crossref PubMed Google Scholar, 21Srivastava M. Pollard H.B. FASEB J. 1999; 13: 1911-1922Crossref PubMed Scopus (430) Google Scholar). To understand telomerase RNP assembly, we wished to identify proteins that regulate hTERT nucleolar localization. Here we show that nucleolin interacts with hTERT in a manner dependent upon hTERC and that this interaction plays an important role in regulating the nucleolar localization of telomerase. Plasmids—The bacterial and mammalian expression vectors for full sized nucleolin and nucleolin mutants as well as the mammalian expression vectors pNKZFLAG-hTERT (amino-terminal FLAG-tagged hTERT), pNCZFLAG-hTERT (carboxyl-terminal FLAG-tagged hTERT), and pNKZGST-hTERT (amino-terminal GST-fused hTERT) have been described previously (22Murakami S. Cheong J.H. Kaneko S. J. Biol. Chem. 1994; 269: 15118-15123Abstract Full Text PDF PubMed Google Scholar, 23Yang T.H. Tsai W.H. Lee Y.M. Lei H.Y. Lai M.Y. Chen D.S. Yeh N.H. Lee S.C. Mol. Cell. Biol. 1994; 14: 6068-6074Crossref PubMed Scopus (103) Google Scholar, 24Hirano M. Kaneko S. Yamashita T. Luo H. Qin W. Shirota Y. Nomura T. Kobayashi K. Murakami S. J. Biol. Chem. 2003; 278: 5109-5115Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 25Arai K. Masutomi K. Khurts S. Kaneko S. Kobayashi K. Murakami S. J. Biol. Chem. 2002; 277: 8538-8544Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We generated an amino-terminal enhanced green fluorescence protein (EGFP)-hTERT fusion protein by replacing the sequence encoding the FLAG epitope in pNKZFLAG-hTERT with EGFP cDNA (pNKZEGFP-hTERT). EGFP cDNA was obtained by PCR using pLEGFP-C1 (BD Biosciences, Clontech) as a template. pGRN164 vector containing hTERC cDNA was linearized with FspI and used as a template for hTERC RNA preparation as described previously (5Masutomi K. Kaneko S. Hayashi N. Yamashita T. Shirota Y. Kobayashi K. Murakami S. J. Biol. Chem. 2000; 275: 22568-22573Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 25Arai K. Masutomi K. Khurts S. Kaneko S. Kobayashi K. Murakami S. J. Biol. Chem. 2002; 277: 8538-8544Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Expression and Purification of Recombinant Proteins—GST-nucleolin fusion proteins were expressed and purified as described previously (26Yamashita T. Kaneko S. Shirota Y. Qin W. Nomura T. Kobayashi K. Murakami S. J. Biol. Chem. 1998; 273: 15479-15486Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Briefly, the transformed bacterial cells (BL21) were harvested by centrifugation and suspended in buffer A (phosphate-buffered saline (PBS) (-), 0.5% Triton X-100, 1 mm dithiothreitol). After centrifugation of the sonicated lysates, the supernatants were passed through DEAESepharose, and the GST fusion proteins were recovered using glutathione-Sepharose 4B beads (Amersham Biosciences). The resin was washed, and the GST fusion proteins were then eluted with glutathione. The eluted solution was dialyzed against buffer B (100 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1 mm dithiothreitol) for 12 h. Amino-terminally FLAG-tagged, recombinant hTERT was produced using baculovirus expression system in High5 insect cells as described previously (5Masutomi K. Kaneko S. Hayashi N. Yamashita T. Shirota Y. Kobayashi K. Murakami S. J. Biol. Chem. 2000; 275: 22568-22573Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Preparation of Cell Extracts, Immunoprecipitation, and Immunoblotting—Cells were harvested, washed with PBS (-), and sonicated in a lysis buffer (50 mm Tris-HCl (pH 7.4), 200 mm NaCl, 1 mm EDTA, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 mm leupeptin, 10 mm aprotinin, 1 mm dithiothreitol). Lysates derived from 5 × 106 cells were diluted 10-fold in the lysis buffer containing 1% bovine serum albumin and precleared by incubation with GammaBind G-Sepharose (Amersham Biosciences) at 4 °C for 1 h. Precleared lysates were incubated with 10 μl of GammaBind G resin with prebound α-FLAG M2 (Sigma) or α-GST (Z-5, Santa Cruz) antibodies at 4 °C for 4 h. Resins were preblocked in lysis buffer containing 1% bovine serum albumin. After an extensive washing (50 mm Tris-HCl (pH 7.4), 300 mm NaCl, 1 mm EDTA, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 mm leupeptin, 10 mm aprotinin, 1 mm dithiothreitol), the bound proteins were fractionated by SDS-PAGE, transferred onto nitrocellulose membranes, and subjected to immunoblot analysis with monoclonal antibodies specific for GST (B-14, Santa Cruz) or the FLAG epitope (M2). For RNase treatment, the same lysates were divided into two tubes and incubated at room temperature for 15 min in the presence or absence of 0.1 μg/μl RNase A followed by the addition of affinity resins. For in vitro GST pull-down experiments, 260 ng of recombinant GST-nucleolin fusion protein was mixed with 220 ng of recombinant FLAG-hTERT in the presence or absence of 250 ng of in vitro transcribed hTERC in a binding buffer containing 1% bovine serum albumin and 250 ng/ml yeast total RNA. The bound proteins were fractionated by SDS-PAGE, transferred onto nitrocellulose membranes, and subjected to immunoblot analysis with α-GST (B-14) or α-FLAG M2 antibodies. Cell Culture and Transient Transfection—IMR90 (normal human lung fibroblasts), Huh7, HLE (hepatoma cell lines), VA13 (SV40-transformed lung fibroblast, subline 2RA) and VA13+hTERC (VA13 cells with stable expression of hTERC) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum (CELLect R GOLD) and 20 μg/ml antibiotics (ampicillin and kanamycin; Meigi Co., Ltd.) and maintained in an incubator with 5% CO2 at 37 °C. Cells were transfected using FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's recommendations. Immunofluorescence and Confocal Microscopy—Cells grown on glass slides were washed once in PBS (-) and fixed with 4% paraformaldehyde in PBS (-) for 20 min. Fixed cells were permeabilized by 0.5% Triton X-100 at room temperature for 10 min and blocked with 1% bovine serum albumin in PBS (-) for 1 h. Cells were then incubated with primary antibody in a humidifying chamber for 1 h, washed three times with PBS (-), and incubated with secondary antibody for 1 h. Subsequently, the cells were washed five times in PBS (-) and mounted using Vectashield mounting medium. Immunofluorescent images were acquired using a Confocal Laser Scanning Microscope (LSM510; Carl Zeiss Co., Ltd). To visualize endogenous nucleolin or GST fusion proteins, cells were stained with a rabbit polyclonal α-nucleolin antibody (1:500 (24Hirano M. Kaneko S. Yamashita T. Luo H. Qin W. Shirota Y. Nomura T. Kobayashi K. Murakami S. J. Biol. Chem. 2003; 278: 5109-5115Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar)) or a rabbit polyclonal α-GST antibody (1:300, Z-5), respectively and visualized with a rhodamine-conjugated goat α-rabbit IgG (1:100, Pierce). Telomerase Activity Assays and Immunoprecipitation-Telomerase Repeat Amplification Protocol (TRAP)—Total lysates of cells were subjected to TRAP assay using a TRAPEZE kit (Intergen) according to the manufacturer's method. For immunoprecipitation-TRAP, lysates from ∼2 × 105 cells/test tube were subjected to immunoprecipitation with a mouse monoclonal antibody specific for hTERT (2C4, subtype IgM (27Masutomi K. Yu E.Y. Khurts S. Ben-Porath I. Currier J.L. Metz G.B. Brooks M.W. Kaneko S. Murakami S. DeCaprio J.A. Weinberg R.A. Stewart S.A. Hahn W.C. Cell. 2003; 114: 241-253Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar)), with a rabbit polyclonal antibody specific for the amino terminus of nucleolin, or with an α-FLAG M2 antibody. As a control, lysates were immunoprecipitated with an isotype-matched, mouse monoclonal antibody specific for α-actinin (Sigma, clone BM-75.2, subtype IgM) or rabbit preimmune serum. The IgG subtype antibodies were bound directly to 10 μl of GammaBind G-Sepharose. The IgM subtype antibodies were bound to same amount of resin using rabbit polyclonal anti-mouse IgM antibody, (Pierce). Telomerase activity was detected using 1 μl of post-immunoprecipitation resin. For in vitro reconstitution assays, for one test tube 100 ng of purified FLAG-hTERT, 200 ng in vitro transcribed hTERC, and increasing amounts of purified nucleolin-1234R were mixed in 20 μl of reconstitution buffer (5Masutomi K. Kaneko S. Hayashi N. Yamashita T. Shirota Y. Kobayashi K. Murakami S. J. Biol. Chem. 2000; 275: 22568-22573Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). After incubation at 33 °C for 10 min, samples were subjected to telomerase reaction using Telochaser kit (Toyobo). Subcellular Localization of hTERT—To establish the subcellular localization of hTERT, we transiently expressed an EGFP-hTERT fusion protein in normal human lung fibroblasts (IMR90) and in a hepatoma-derived cancer cell line (Huh7). Ectopic expression of EGFP-hTERT conferred telomerase activity in IMR90 cells as well as a slight increase of telomerase activity in Huh7 cells (Fig. 1A), indicating that EGFP-hTERT formed an active telomerase complex. IMR90 cells expressing EGFP-hTERT showed a pan-nuclear localization of EGFP-hTERT with enriched nucleolar localization as assessed by costaining with an anti-nucleolin-specific antibody. In contrast, Huh7 cells expressing EGFP-hTERT exhibited a diffuse nucleoplasmic distribution of EGFP-hTERT with exclusion of EGFP-hTERT from the nucleoli (Fig. 1B), consistent with a prior report (11Wong J.M. Kusdra L. Collins K. Nat. Cell Biol. 2002; 4: 731-736Crossref PubMed Scopus (216) Google Scholar). We note that although the percentage of EGFP-hTERT-expressing cells in the transfected population of IMR90 cells was lower than those in Huh7 cells (20 and 60% of the cell population, respectively), the distinct localization pattern of EGFP-hTERT between these cell lines was reproducible. It may be related to a lower expression level of hTERC in normal cells than that in transformed cells (data not shown) or reflect deregulation of shuttling process of hTERT between the nucleolus and nucleoplasm in transformed cells (see "Discussion"). Interactions between hTERT and Nucleolin—The colocalization of EGFP-hTERT with nucleolin in nucleoli of normal not in cancer that nucleolin and hTERT To this GST-fused hTERT was transiently expressed in IMR90 or Huh7 cells. we used an α-GST antibody to hTERT from IMR90 we endogenous nucleolin in these (Fig. the nucleoplasmic localization of hTERT, endogenous nucleolin was also detected in derived from Huh7 cells expressing (Fig. nucleolin was recovered in α-FLAG from IMR90 cells and Huh7 cells expressing FLAG-tagged hTERT (data not A that interaction of hTERT and nucleolin occurs not in cells in cell is recombinant proteins in lysates not with the interaction of the two protein These indicate that the interaction of hTERT and nucleolin occurs in normal and cancer that the interaction of nucleolin and hTERT occurs in the nucleolus and in the the assembly of hTERT with hTERC may in the nucleolus to other RNPs (11Wong J.M. Kusdra L. Collins K. Nat. Cell Biol. 2002; 4: 731-736Crossref PubMed Scopus (216) Google Scholar, 12Yang Y. Chen Y. Zhang C. Huang H. Weissman S.M. Exp. Cell Res. 2002; 277: 201-209Crossref PubMed Scopus (97) Google Scholar, 13Etheridge K.T. Banik S.S. Armbruster B.N. Zhu Y. Terns R.M. Terns M.P. Counter C.M. J. Biol. Chem. 2002; 277: 24764-24770Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, M.O. Dundr M. Szebeni A. Trends Cell Biol. 2000; 10: 189-196Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 15Pederson T. Politz J.C. J. Cell Biol. 2000; 148: 1091-1095Crossref PubMed Scopus (102) Google Scholar), we that the binding of hTERT with nucleolin the presence of hTERC. we total cell lysates with RNase we that this not the amount of nucleolin recovered in the in normal and cancer cells (Fig. These findings that one or RNA to the interaction between nucleolin and hTERT. To the specific interaction between nucleolin and hTERT, we the of nucleolin for its interaction with hTERT. For these experiments, we transiently FLAG nucleolin or a of mutants (Fig. with a fusion protein into Huh7 cells. an α-GST antibody, we that nucleolin and two nucleolin nucleolin-1234R and bound In contrast, mutants containing the amino terminus of nucleolin and RGG domain to (Fig. the binding of to hTERT was by RNase A (Fig. and These indicate that nucleolin two for hTERT The binds through protein-protein the the of RNA. RNase A the amount of hTERT recovered in the containing full sized nucleolin or is consistent with the that protein-protein and interactions to the binding of hTERT and of hTERT and Nucleolin in the binding of nucleolin and hTERT was we purified bacterial GST-nucleolin proteins, purified insect FLAG-hTERT and purified hTERC and these purified proteins hTERT in the presence and absence of hTERC (Fig. and not or GST bound hTERT (Fig. The interaction of hTERT and nucleolin was detected with nucleolin-1234R and (Fig. and The for the hTERT binding was consistent with the analysis (Fig. also bound hTERT in although this interaction was than that with the telomerase (Fig. and RNase A of total cell lysates the interaction between and hTERT (Fig. may be of the of amounts of the purified proteins in vitro than those in the mammalian cell may hTERT binding The of hTERC on the binding between hTERT and nucleolin in vitro (Fig. that hTERC is a major RNA that to the interaction between nucleolin and telomerase. The of hTERC on the binding between hTERT and not may indicate that the is the hTERC binding these indicate that the specific interaction of telomerase and nucleolin and the RGG of nucleolin and that hTERC is in this may also to the binding of telomerase with nucleolin through interactions with hTERC (Fig. of human nucleolin and its with of binding to hTERT. The binding of to hTERT in the presence or absence of hTERC is on the binding not Telomerase Activity in Nucleolin endogenous nucleolin and telomerase we immunoprecipitation with an α-nucleolin antibody and that we telomerase activity in these (Fig. A and we using cells expressing the and which are to hTERT, we to telomerase activity (Fig. that of telomerase activity upon the interaction between telomerase and These findings the that endogenous nucleolin, at in interacts with the telomerase complex. these indicate that the interaction of nucleolin with hTERT not telomerase To nucleolin telomerase we an in vitro telomerase activity The addition of purified recombinant human nucleolin-1234R to or in vitro telomerase activity was in to hTERT (Fig. These that nucleolin not telomerase enzymatic Nucleolin Subcellular Localization of nucleolin and hTERT and we nucleolin the subcellular localization of hTERT. and of nucleolin were expressed as GST fusion proteins in Huh7 cells expressing EGFP-hTERT (Fig. Although we diffuse in the nucleoplasm, of full sized nucleolin in enriched localization of EGFP-hTERT in nucleoli (Fig. In contrast, of the of nucleolin which binds hTERT, with EGFP-hTERT showed distribution (Fig. of the with EGFP-hTERT to the localization of EGFP-hTERT (Fig. these that nucleolin the subcellular localization of hTERT. To these we subcellular localization of EGFP-hTERT using VA13 which telomeres through an than through the expression of telomerase. cell line hTERC and hTERT T.M. L. S. M. R.R. Mol. Genet. 1997; PubMed Scopus Google Scholar, C. M.A. S. 2001; PubMed Scopus Google Scholar). expression of EGFP-hTERT in VA13 cells in nucleolar localization of EGFP-hTERT in the absence of hTERC. The of hTERC this localization of EGFP-hTERT to the nucleoplasm (Fig. EGFP-hTERT expression in telomerase activity in VA13+hTERC although VA13 cells to show telomerase activity upon expressing of EGFP-hTERT (Fig. FLAG-hTERT derived from VA13 and in VA13+hTERC endogenous nucleolin (Fig. the show that the interaction of nucleolin with hTERT and hTERC the subcellular localization of telomerase. The of subcellular localization of hTERT, the catalytic subunit of the telomerase may regulate the of telomerase by of the telomerase complex to telomeres or by the of telomerase RNP Several that nucleoli and/or are the for telomerase RNP assembly hTERT and hTERC are in these (11Wong J.M. Kusdra L. Collins K. Nat. Cell Biol. 2002; 4: 731-736Crossref PubMed Scopus (216) Google Scholar, 12Yang Y. Chen Y. Zhang C. Huang H. Weissman S.M. Exp. Cell Res. 2002; 277: 201-209Crossref PubMed Scopus (97) Google Scholar, 13Etheridge K.T. Banik S.S. Armbruster B.N. Zhu Y. Terns R.M. Terns M.P. Counter C.M. J. Biol. Chem. 2002; 277: 24764-24770Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 16Mitchell J.R. Cheng J. Collins K. Mol. Cell. Biol. 1999; 19: 567-576Crossref PubMed Scopus (437) Google Scholar, 17Narayanan A. Lukowiak A. Jady B.E. Dragon F. Kiss T. Terns R.M. Terns M.P. EMBO J. 1999; 18: 5120-5130Crossref PubMed Scopus (109) Google Scholar, 18Lukowiak A.A. Narayanan A. Li Z.H. Terns R.M. Terns M.P. RNA (N. Y.). 2001; 7: 1833-1844PubMed Google Scholar, Y. R.L. Lukowiak A.A. Terns R.M. Terns M.P. Mol. Biol. Cell. PubMed Scopus Google Scholar, J. Genes Dev. 18: PubMed Scopus Google Scholar, B.E. E. Kiss T. J. Cell Biol. PubMed Scopus Google Scholar, J.L. Greider C.W. Trends Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). a report showed that hTERT distribution between nucleoli and the nucleoplasm is in a cell manner in normal and deregulation of the localization of hTERT in cancer cells with the in transformed cells (11Wong J.M. Kusdra L. Collins K. Nat. Cell Biol. 2002; 4: 731-736Crossref PubMed Scopus (216) Google Scholar). In this report we that nucleolin binds the active telomerase complex through protein-protein and distinct of nucleolin are in the interaction with a containing the and a carboxyl-terminal containing the and RGG It the binds hTERC or also interacts with hTERT in telomerase. Although other protein-protein or interactions may also these the indicate that nucleolin is a binding for telomerase. Although the interaction of nucleolin and hTERT to telomerase activity in this interaction the subcellular localization of hTERT. In this these findings are of a prior report that that the interaction of nucleolin and a RNA-dependent RNA the subcellular localization of (24Hirano M. Kaneko S. Yamashita T. Luo H. Qin W. Shirota Y. Nomura T. Kobayashi K. Murakami S. J. Biol. Chem. 2003; 278: 5109-5115Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). the interaction of hTERT and nucleolin, the interaction of nucleolin with activity (24Hirano M. Kaneko S. Yamashita T. Luo H. Qin W. Shirota Y. Nomura T. Kobayashi K. Murakami S. J. Biol. Chem. 2003; 278: 5109-5115Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We show that nucleolin subcellular localization of hTERT the hTERT binding the subcellular localization of hTERT in colocalizes with in the the a localization this protein-protein interaction of hTERT and is critical for the subcellular localization of telomerase. the distinct hTERT subcellular localization between normal and transformed expressed hTERT to with endogenous nucleolin from of These that the interaction between nucleolin and telomerase occurs not in nucleoli also in the for these findings is that of nucleolin or hTERT may nucleoplasmic localization of hTERT in cancer cells. other proteins may the localization of the complex in normal cells and cancer cells. In nucleolin to be in assembly or of telomerase subcellular localization of hTERT was by the presence or the absence of hTERC as in VA13 cells. The nucleolar localization of hTERT in VA13 cells was not an of cells in cell which hTERC, hTERT was to be to the nucleoplasm and not of EGFP-hTERT and also localization of EGFP-hTERT to the in VA13 cells as was in the Huh7 cells. In contrast, the nucleolar in VA13 cells was by expression of The that the of nucleolin and the hTERT complex was by the presence of hTERC. Nucleolin as an RNA chaperone with hTERT may assembly of telomerase in the presence of hTERC. are then from nucleoli to the nucleoplasm in a process that may of a nucleolar of hTERT and/or factors to regulate this process of full sized nucleolin in nucleolar colocalization of telomerase in cancer cells. The of the of nucleolin with telomerase in the nucleoplasm is that nucleolin telomerase in the nucleoplasm for the delivery to telomeres. Nucleolin not telomerase also a of telomerase for delivery to the telomerase is in compartments in normal and human these that in telomerase assembly and localization play additional roles in cell We J. W. Shay for VA13 and VA13+hTERC cells and of the for and We F. K. and M. for with