Nucleolar Localization of hTERT Protein Is Associated with Telomerase Function

Experimental Cell Research 277, 201–209 (2002) doi:10.1006/excr.2002.5541

Nucleolar Localization of hTERT Protein Is Associated with Telomerase Function Yinhua Yang, 1 Yaohui Chen, 1 Chunyu Zhang, Hai Huang, and Sherman M. Weissman 2 Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06536-0812

Telomerase is a ribonucleoprotein (RNP) complex that prevents telomeric erosion in eukaryotic cells. Although there are also other associated proteins in this complex, the catalytic activity of this complex is composed of two components. One is a reverse transcriptase subunit, TERT (telomerase reverse transcriptase); another is an RNA template subunit, TR (telomerase RNA). However, where these two parts are assembled in mammalian cells is unclear. In the present study, we investigated the intracellular distribution of human TERT (hTERT) protein and observed that hTERT protein in individual cells could concentrate in or be excluded from the nucleolus. Further we have identified a nucleolar targeting signal in the hTERT protein. Point mutations that disrupted this signal region interrupted telomerase RNP complex formation, decreased telomerase activity, and caused telomere shortening in cells transfected with mutated hTERT. Our results indicate that the amino acid sequence of the extreme N-terminus (1–15) of hTERT, which targets nucleolar localization of the protein, is required for full telomerase function. © 2002 Elsevier Science (USA)

Key Words: human; telomerase; hTERT; hTR; nucleoli.

INTRODUCTION

Telomeres are the extreme ends of a eukaryotic chromosome and protect the chromosomal ends from degradation, rearrangement, and fusion events. In humans, telomeres are made up of about 5–15 kb of {TTAGGG} repeats and telomere-binding proteins (for review see [1]). During each cell division, telomeres lose 50 –100 bp due to the “end-replication problem” [2]. When a telomere loses a critical number of base pairs, it triggers a signal for the cell to stop dividing and induce senescence [3]. In eukaryotes, cells have evolved strategies to counteract this progressive telomere loss. Among these, elongation of telomeres by the 1

These authors contributed equally to this work. To whom correspondence and reprint requests should be addressed. Fax: (203) 737-2286. E-mail: [email protected]. 2

reverse transcriptase activity of telomerase is the most common and well-studied mechanism for telomere length maintenance. Telomerase, a ribonucleoprotein (RNP) complex, prevents telomeric shortening by adding TTAGGG repeats to the 3⬘ end of telomeres. This RNP complex is composed of two core components, an RNA template [telomerase RNA, (TR)] subunit and a reverse transcriptase [telomerase reverse transcriptase (TERT)] subunit. Associated proteins are also found in the complex (for a review see [4]). Human TR subunit (hTR) has 451 nucleotides. Within this RNA, nucleotides 46 –56 serve as a binding site for telomere ends and as a template for the addition of telomeric repeats. A conserved H/ACA box is found in the 3⬘ portion of hTR and is responsible for hTR targeting to the nucleolus and necessary for hTR accumulation [5, 6, 35]. The hTR subunit ubiquitously exists in both normal and malignant tissues [7, 8]. Human TERT subunit (hTERT) is a protein of 1132 amino acids with a highly conserved domain homologous to reverse transcriptase (RT). This RT domain is located towards the C-terminal region of the protein [9, 10]. Recently, four conserved motifs, GQ, CP, QFP and T, were identified in the N-terminal region of all TERTs [11] and the function of these domains of human telomerase has been studied in detail [12]. Research on Tetrahymena and human telomerase showed that T and CP motifs outside the RT domain provided the major interactions with the RNA subunit [12–15]. In contrast to hTR, hTERT is only expressed in a limited number of normal tissues, such as germ cells, stem cells, and activated lymphocytes, but is highly expressed in immortalized cells and most tumor tissues [9, 16]. Telomerase activation requires the interaction between TERT and TR. In vitro studies showed that telomerase activity could be reconstituted using only hTR and hTERT although a variety of additional factors may participate in regulating or promoting in vivo activity [17–20]. Mouse fibroblast cells lacking the RNA component of telomerase exhibited progressive telomere shortening and chromosomal instability [21].

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0014-4827/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

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Late generations of TR knockout mice had shorter telomeres and an increased incidence of spontaneous tumors [22]. The cells from dyskeratosis congenita patients have markedly shorter telomeres than those from normal individuals. Recently, evidence was found that some of these patients had mutations in the hTR gene that caused defective telomerase activity [23]. All these data show that the combination of both subunits is necessary for telomerase function. However, where and exactly how these two parts are assembled to form the active RNP complex in mammalian cells remains unknown. We report here that human TERT protein could concentrate in or be excluded from the nucleolus. A nucleolar targeting signal of hTERT protein has been identified. Point mutations in this signal region interrupted telomerase RNP complex formation, decreased telomerase activity, and caused telomere shortening in cells transfected with the mutated hTERT. MATERIAL AND METHODS Plasmids Green fluorescence protein- (GFP) tagged full-length hTERT. All pEGFP vectors were purchased from Clontech. The telomerase catalytic subunit expressing vectors pCI-Neo-hTERT-HA (GenBank Accession No. AF043739) and pCI-Neo-hTERT were kindly provided by Dr. Robert A. Weinberg [24]. The EcoRI–SalI fragment containing the full-length hTERT cDNA (GenBank Accession No. AF018167) was cloned into pEGFP-C2 vector. GFP-tagged hTERT fragments. hTERT 1– 686 (EcoRI–EcoRV) was subcloned into the EcoRI–SmaI site of pEGFP-N2. hTERT 656 – 1132 (XhoI–SalI) was subcloned into pEGFP-C2. hTERT 1– 471 (EcoRI–NaeI), hTERT 1–219 (EcoRI–SmaI), and hTERT 1–114 (EcoRI–AatI) were subcloned into the EcoRI–SmaI sites of pEGFP-N1, -N3, and -N1, respectively. hTERT 1–33 (EcoRI–ApaI) was subcloned into the EcoRI–ApaI site of pEGF-N1 vector. hTERT 1–15 was amplified by PCR using the primer P RI (5⬘-GAATTCGTCCTGCTGCGCA-3⬘) and the primer P HI (5⬘-GGATCCGCAGCAGGGAGC3⬘) and subcloned between the EcoRI and BamHI sites of pEGFP-N2. To make the hTERT 16 –1132 construct, we first PCR amplified a fragment containing EcoRI and MluI sites, but without the first 15 amino acid encoding region, with the primer P Tf (5⬘-TAGAATTCACTACCGCGAGGTG-3⬘) and the primer P Tr (5⬘-ACACAGAAACCACGGTCACT-3⬘) and then we replaced the pBKS(⫺)hTERT EcoRI–MluI part of hTERT by the PCR product to generate pBKS(⫺)-hTERT161132. Finally, we subcloned the EcoRI–SalI fragment from pBKS(⫺)hTERT16-1132 into pEGFP-C3. GFP-tagged point-mutated hTERT. Point mutations were generated by PCR amplification of the 5⬘ end of hTERT cDNA with mutation creating primers. Mut-1 changed the first arginine (AA-3) to alanine, Mut-2 changed the first two arginines (AA-3 and AA-6) to alanines, Mut-4 changed the first four arginines (AA-3, AA-6, AA-8, and AA-11) to alanines, Mut-5 and Mut-6 changed the third (AA-8) and the fourth (AA-11) arginine to alanine respectively, and Mut-7 is the combination of Mut-5 and Mut-6 (AA-8 and AA-11). We inserted the point-mutated hTERT into pEGFP-N2 vector between the EcoRI and XhoI sites. All the constructs were verified by DNA sequencing. The EcoRI–SalI fragment that encoded a full-length catalytically inactive, dominant negative form of hTERT in which aspartic acid and valine residues at positions 710 and 711 were replaced respectively with alanine and isoleucine, derived from pCIneo-DN-hTERT (a gift from Dr. Weinberg) [27], was inserted into the pEGFP vector to form pEGFP-DN-hTERT.

Cell Culture and Transfection Hela cells and 293 cells were purchased from ATCC. GM847 cells were from Coriell Medical Research Institute. Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (GIBCO/BRL). Cells were transfected with the indicated constructs by use of the FuGENE6 reagent (Roche) according to the manufacturer’s instruction. Stable expression cell lines were established by selection with 500 ␮g/ml G418 (GIBECO/BRL) for 2 weeks. Cells for telomere length measurement were passed every 3 days until 36 passages were run. Immunostaining Cells were washed twice with phosphate-buffered saline (PBS) and fixed in methanol/acetone (1:1) at ⫺20°C for 20 min. The coverslips were air dried and then incubated with rabbit anti-hTERT polyclonal antibody (Calbiochem) (1:200) or with mouse antinucleolin monoclonal antibody (MBL) (1:200) for 1 h at 37°C, followed by exposure to appropriate secondary antibodies conjugated to Texas Red or fluorescent isothiocyanate (FITC) (Jackson) (1:200). The coverslips were mounted in VECTASHIELD medium with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Vector Laboratories) and then observed and photographed with a Nikon Eclipse E800 immunofluorecent microscope. Isolation of Nucleoplasm and Nucleolar Proteins The method was the same as we previously described [25]. Briefly, cells were swelled for 20 min on ice in lysis buffer [10 mM Tris–HCl (pH 7.4) 10 mM KCl, 2 mM MgCl 2, 0.05% Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM 1,10phenanthroline, 1 mM ethyleneglycol-bis-(␤-aminoethylether)N,N,N⬘,N⬘-tetraacetic acid, 10 mM E64, and protease inhibitors (Roche)]. A crude nuclear pellet was isolated by forcing the cell suspension through a 27-gauge needle and centrifuging for 5 min at 700g. Nuclei were resuspended in lysis buffer containing 10 mM KCl, disrupted by 15 pulse sonication, and centrifuged at 5,000g for 5 min. The resulting supernatants contained mostly nucleoplasmic proteins, whereas pellets were highly enriched with nucleoli. The pellets were extracted again in lysis buffer containing 300 mM KCl for 45 min on ice and separated into supernatants and pellets by centrifugation at 16,000g. The resulting supernatants contained nucleolar proteins. Northern and Western Blots GM847 cells were stably transfected with the indicated constructs, and total RNA and protein were isolated from the cells by standard methods. hTERT probe, hTR probe, and GAPDH probe were labeled with [ 32P]␣-dCTP by polymerase chain reaction (PCR) amplification of the correspondent cDNAs. The sequences of PCR primers are 5⬘-CGGGCCTGGAACCATAGC-3⬘ and 5⬘-CTTGAAGGCCTTGCGGACGT-3⬘ for hTERT; 5⬘-CACGAGAGCCGCGAGAGTCA-3⬘ and 5⬘TGTGAGCCGAGTCCTGGGTG-3⬘ for hTR; and 5⬘-GGTCATCCCTGAGCTGAACG-3⬘ and 5⬘-GATGGTACATGACAAGGTGC-3⬘ for GAPDH. The Northern blot was hybridized in Church–Gilbert buffer at 65°C overnight. Proteins from each transfected group were electrophoresed on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to a nitrocellulose membrane. The protein expression levels were detected by anti-GFP monoclonal antibody (Roche). IP–Northern Blot Protein G–Sepharose beads (Pharmacia Biotech) were incubated with anti-GFP monoclonal antibody in IP buffer [20 mM Tris–HCl (pH 7.4) 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.65% NP-40] at 4°C for 1 h. After washing three times with

NUCLEOLAR LOCALIZATION OF hTERT IS CORRELATED WITH FUNCTION OF hTERT IP buffer, the antibody-coated beads were incubated with nuclear and nucleolar proteins mentioned above in IP buffer containing RNase inhibitor (Roche) at 4°C for overnight. The beads were recovered by brief centrifugation. The supernatants were transferred to clean fresh tubes, and the beads washed three times and resuspended with IP buffer and then proteins were extracted from the supernatants and the beads with phenol/chloroform once and then with chloroform once. The RNAs in the aqueous phase were precipitated by ethanol. Precipitated RNAs were resuspended in 10 ␮l diethy pyrocarbonate-treated H 2O and subjected to Northern blotting. TR probe was labeled with [ 32P]␣-dCTP by PCR and the Northern blot was hybridized in Church–Gilbert buffer at 65°C overnight. The membrane was washed with 2⫻ SSC/0.1% SDS briefly, 1⫻ SSC/0.1% SDS for 30 min, and 0.1⫻ SSC/0.1% SDS for 30 min at 65°C and exposed to a film for a few days at ⫺20°C. TRAP Assay Nuclear proteins were prepared as described above. The concentration of nucleoplasmic and nucleolar proteins was determined by the Bradford method (Bio-Rad). The TRAP assay was performed by use of Telo TAGGG Telomerase PCR ELISAPLUS kit (Roche). A TRAPEZE Telomerase Detection kit (Intergen) was also used according to the protocol provided by the manufacture. Telomere Length Measurement Genomic DNAs were isolated from stably transfected cells using cell lysis buffer (100 mM Tris–HCl, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, and 400 ␮g/ml Proteinase K) and precipitated by 2-propanol. The DNAs were digested with restriction enzymes RsaI and HinfII at 37°C overnight and then electrophoresed in 1% agarose gel. After denaturation and neutralization, the DNAs were transferred to Hybond-N⫹ nylon membrane and fixed by UV crosslinking. Telomeric probe (CCCTAA) 4 was labeled with [ 32P]␥-ATP by T 4 polynucleotide kinase. Prehybridization and hybridization were performed at 58°C overnight. The membrane was washed with solution 1 (3⫻ SSC, 10 mM sodium phosphate, 10⫻ Denhart’s solution, and 5% SDS) and solution 2 (1⫻ SSC and 1% SDS) sequentially at 65°C for 2 h and then exposed to film at ⫺20°C for 5 days.

RESULTS

hTERT Protein Intracellular Distribution To investigate hTERT protein intracellular localization, GFP was fused to the N-terminus of hTERT and the tagged TERT construct was stably transfected into TERT-negative/TR-positive GM847 cells. In addition to cytoplasmic localization, three distribution patterns of hTERT-GFP fusion protein were observed in the nuclei of an asynchronous cell population. Most cells showed a whole nuclear distribution pattern (⬃74%), a small portion of cells presented a nucleolar exclusion pattern (⬃21%), and a few cells (⬃5%) exhibited a nucleolar concentration pattern (Fig. 1A). The cells with TERT concentrated in the nucleolus usually appeared in twoto eight-cell clusters, which suggests that hTERT might pass through the nucleolus at a specific stage in a subgroup of cells (Fig. 1B). Since there is no endogenous hTERT protein in GM847 cells, we wondered whether the endogenous hTERT protein has the same distribution patterns in mammalian cells. Therefore, we used anti-hTERT

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polyclonal antibody to detect the endogenous hTERT protein in telomerase-positive Hela cells. Similar phenomena were observed in Hela cells with 84% showing a nuclear distribution pattern, 10% a nucleolar exclusion pattern, and 6% a nucleolar concentration pattern (Fig. 1C). Because the nucleolar localization of hTERT protein has not been reported before, we used antinucleolin monoclonal antibody to colocalize hTERT protein with nucleolin, a nucleolar protein, to confirm hTERT localization. The two signals merged together, which indicates that the hTERT protein does exist in the nucleolus in the cells and exhibits a nucleolar concentration pattern (Fig. 1C). The nucleolar concentration and exclusion patterns of hTERT protein were also observed in 293 cells (hTERT and hTR positive) and in stably transfected SV40-WI38 cells (hTERT and hTR negative) (data not shown). To further study whether there is a relationship between the nucleolar translocalization and cell cycle, we first observed hTERT-GFP protein distribution in stably transfected GM847 or SV40-WI38 cells at different time points after release from 72 h of serum starvation. Cells with each of the three distribution patterns were counted at 9 time points (0, 12, 20, 24, 30, 34, 36, 38, and 40 h). At the 0-, 12-, and 20-h points, the green signal was very weak and distributed in the nucleus. At 24 h, the nucleolar concentration and exclusion patterns began to be seen in a few cells (⬃5%). Around 34 h, the green signal became stronger, and the nucleolar concentration pattern could be observed in more cells (⬃28%). At 40 h, the nucleolar concentrated cells decreased to ⬃3%. In this experiment S phase occurred around 30 h after release from 72 h serum starvation, as judged by a standard BrdU incorporation assay (unpublished data). The experiment was repeated several times, and also with HeLa cells. The peak nucleolar concentration was consistently observed at between 24 and 38 h after refeeding of cells, while the peak of BrdU incorporation in three of four experiments occurred about 2 h earlier. On average 30% of cells were labeled after 30-min exposure to BrdU at the peak of the S phase using a BrdU labeling and detection kit (Roche). Further studies are needed to investigate the relationship between nucleolar localization of TERT and cell-cycle progression. A Nucleolar Localization Signal of hTERT Protein Is at the Extreme N-Terminus If the hTERT protein can enter into the nucleolus, there may exist nucleolar localization signal(s) in this protein. To identify potential nucleolar targeting signal(s), GFP protein was fused to different fragments of hTERT and the fusion protein expression vectors were introduced into GM847 cells (Fig. 2). We observed that the C-terminus (residues 656 –1132) of hTERT, which

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FIG. 1. Intracellular localization of hTERT protein. (A) Ectopic GFP-hTERT fusion protein in GM 847 stable expression cells showed three distribution patterns in the nucleus: (1) a nuclear distribution pattern, (2) a nucleolar concentration pattern, and (3) a nucleolar exclusion pattern. The upper row of images shows the GFP fluorescence; the lower row is phase-contrast imaging of the same cells. (B) The cells with a nucleolar concentration pattern appeared in clusters. (C) The endogenous hTERT protein distribution in Hela cells. In the upper panel, the endogenous hTERT was stained with a rabbit anti-hTERT and FITC-conjugated antirabbit antibody. In the middle panel, endogenous nucleolin was stained with a mouse monoclonal antinucleolin and Texas Red-conjugated antimouse antibody. In the bottom panel, overlay images show the colocalization of hTERT and nucleolin in nucleoli. Endogenous hTERT protein has three distribution patterns in Hela cells: (1) a nuclear distribution pattern, (2) a nucleolar concentration pattern, and (3) a nucleolar exclusion pattern.

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FIG. 2. Nucleolar localization signal region of hTERT protein. (A) Full-length hTERT protein with restriction sites and different domains as described [11, 12]. (B) Different hTERT fragments were tagged with GFP protein and stably introduced into GM847 cells. Three hundred transfected cells were counted for each group. The percentage of the three distribution patterns in each stable expression cell line is shown on the right. The color pictures represent typical patterns: (1) a nuclear distribution pattern, (2) a nucleolar concentration pattern, and (3) a nucleolar exclusion pattern.

contains the A, B⬘, C, D, and E portions of the RT motif and a nuclear export signal-like motif, was homogenously distributed in the nucleus and cytoplasm in all

transfected cells (100%), while the N-terminus of hTERT (residues 1– 686) containing the GQ, CP, QFP, and T motifs as well as RT motifs 1 and 2 showed

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amino acids from the full-length hTERT protein, the nucleolar concentration pattern disappeared. Similar results were obtained with the SV40-WI38 cell line that lacked hTERT and hTR (data not shown). All results together indicated that the first-15-amino-acid domain seems to serve as a nucleolar targeting signal of hTERT protein, and the region between residues 471 and 686 plays a role in the regulation of the process. Point Mutations in the Signal Region Prevent the Protein Entering into the Nucleolus To further investigate the role of the N-terminal nucleolar targeting signal in the nucleolar localization of hTERT, we made point mutations in the first 15amino-acid signal region. We changed the positively charged arginine residues to alanines (MPR/AAPR/ ACR/AAVR/ASLLR) individually or in various combinations in the GFP-tagged N-terminus of hTERT (Fig. 3A) and compared the mutated hTERT intracellular localization with that of the wild-type N-terminus of hTERT. No single point mutation had any significant

FIG. 3. Effects of N-terminal point mutations on nucleolar localization of hTERT protein. (A) Various combinations of the amino terminal four arginines were changed to alanines. (B) The constructs were stably introduced into GM847 cells and the percentage of three distribution patterns was counted in each cell line. (C) Alignment of the N-terminal 15 amino acids of TERT from various species. Bold type indicates conserved residues among TERTs (Homo sapiens: AAC51724; Mus musculus: O70372; Melanochromia auratus: AAF17334; Arabidopsis thaliana: NP_197187; Tetrahymena thermophila: T14891; Oxytrichia trifallax: O76332).

nucleolar concentration pattern in a majority of the cells (61%). When the N-terminal fragment was deleted to 471 amino acids, the nucleolar concentration pattern disappeared and the nucleolar exclusion pattern dramatically increased (81%), which implied that the region between residues 471 and 686 plays a role in modulating nucleolar localization of N-terminus (residues 1– 686) of hTERT protein. The domain (residues 471– 686) seems to not contain nucleoli targeting or an intact exclusion signal because when EGFP-hTERT (471– 686) was stably expressed in GM847 or Hela cells it showed a diffuse nuclear localization pattern (result not shown). With further deletions retaining 219, 114, or 38 amino acids of the N-terminal portion of TERT, the percentage of the cells with a nucleolar concentration pattern increased again and the nucleolar exclusion pattern decreased. The extreme N-terminal fragment containing only the first 15 amino acids (MPRAPRCRAVRSLLR) could still accumulate in the nucleolus (62%). However, when we deleted the first 15

FIG. 4. Effects of WT-hTERT and hTERT mutants on telomerase RNP formation. GM847 cells were stably transfected with pEGFP vector (V) or EGFP fused wild-type hTERT (WT), dominant negative point-mutated hTERT (DN), N-terminal two-point-mutated hTERT (M2) and N-terminal four-point-mutated hTERT (M4). (A) Northern blot showing the expression level of ectopic GFP-TERT (upper panel) and endogenous hTR (middle panel) in each stable expression cell lines. GAPDH (bottom panel) was a sample loading quantity control. (B) Western blot showing the expression levels of ectopic hTERT proteins in each cell lines. (C) GFP-hTERT proteins were immunoprecipitated from each stable expression cell line by anti-GFP antibody. RNAs were extracted from the immunoprecipitates and used for Northern blot analysis. (Upper panel) RNA from IP supernatants (S); (middle and bottom panels) RNA from antibody-bond fractions (B). hTR probe and U64 probe were used to hybridize the RNA components.

NUCLEOLAR LOCALIZATION OF hTERT IS CORRELATED WITH FUNCTION OF hTERT

effects on the hTERT fragment nucleolar location, nor did the double point mutations of the Mut-7 (data not shown). However, the percentage of nucleolar concentration pattern decreased from 62 to 38% in the cells transfected with Mut-2 in which the first two arginine residues mutated to alanines; whereas in the cells transfected with Mut-4 in which all four arginine residues were mutated to alanines, the percentage of nucleolar concentration pattern decreased further to 5% (Fig. 3B). We also compared the Mut-4 full-length hTERT transfected cells with wild-type full-length hTERT transfected cells. The results showed that the Mut-4 full-length mutant did not concentrate in the nucleoli in any cell (Fig. 3B), suggesting that the four positively charged amino acids are required for hTERT entering into the nucleolus. By NCBI-BLAST analysis we observed that the nucleolar targeting signal of TERT is well conserved among human, mouse, golden hamster, and Arabidopsis thaliana (Fig. 3C). Tetrahymena and Oxytrichia telomerase did not contain these arginines but were enriched for other basic amino acids in the N-terminal 15 residues. Therefore we speculate that the TERT proteins of mouse, hamster, Arabidopsis, and possibly Terahymena and Oxytrichia also contain an N-terminal nucleolar localization signal. hTERT Mutants Interrupt RNP Complex Formation Since the TR subunit of telomerase has been reported to localize partly in the nucleolus [5, 26, 35], we hypothesized that hTERT and TR subunits might assemble in the nucleolus to form an active telomerase RNP complex. To test this hypothesis, we used antiGFP monoclonal antibody to immunoprecipitate the nuclear and nucleolar proteins from GM847 cells that were stably transfected with GFP vector (GFP), wildtype GFP-hTERT (WT), dominant negative [27] GFPhTERT (DN), or the N-terminus point-mutated GFPhTERT (M2 and M4). Then we extracted RNAs from the immunoprecipitates and hybridized the Northern blot with hTR probe. The results showed that the telomerase RNA component could be detected in supernatants or in immunoprecipitates of GFP-TERT WT, GFP-TERT DN, and GFP-TERT M2, but the RNA component was barely detected in the antibody-bound fraction from GFP-TERT M4 transfected cells (Fig. 4), indicating that telomerase RNP complex formation had been interrupted by the extreme N-terminal four-point mutations. As a control for immunopurification specificity, we used U64, a small nucleolar RNA with an H/ACA box [5], as a probe to hybridize to the Northern blot. No signal was detected in any of the antibodybound fractions, indicating that the GFP-hTERT fusion protein is specifically binding to hTR.

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Telomerase Loses Its Function in the Cells Transfected with Mutated hTERT Several lines of evidence have proved that RNP complex formation is essential for telomerase function. If the point mutations of hTERT protein can disrupt this process, telomerase function could be affected. To investigate if this was the case, we established GM847 cell lines that stably expressed the wild-type or mutant proteins mentioned above but without GFP tags and measured the telomerase activities of these cell lines by the TRAP assay. As we expected, telomerase activity was not detected in the catalytically inactive, dominant negative hTERT- (DN-hTERT, [27]) transfected cells and slightly decreased in Mut-2-transfected cells, but dramatically decreased in the cells transfected with Mut-4 compared with wild-type hTERT (Figs. 5A and 5B). We found that telomerase activity in N-terminal 15-amino-acid-deleted hTERT-transfected cells was similar to Mut-4 transfected cells (not shown). We also established 293 cell lines (a human embryonic kidney cell line) that stably expressed control vector or wild-type and mutant hTERT proteins. Consistent with our telomerase activity assay, telomere length of the Mut-4 transfected cells was shorter than control groups that expressed empty vector alone at 36 passages, indicating that telomerase function in vivo has also been affected by the point mutations in the nucleolar targeting signal region of hTERT protein (Fig. 5C). DISCUSSION

The nuclear localization of human TERT protein has previously been reported [28, 29]. Our present studies revealed that hTERT protein can enter into or be excluded from the nucleolus and the ability to localize in the nulceolus is associated with active telomerase complex formation, enzyme activity, and biological function. hTERT and hTR combination is essential for telomerase activity, and the interaction domains have been identified for both hTERT and hTR [6, 13, 19, 20, 30, 31]. However how and where the two components assemble in vivo remain to be elucidated. In our study we observed hTERT-GFP protein occurred in different subnuclear compartments in single cells. There are several possibilities for the explanation of this phenomenon. The nucleolus might be involved in prevention of the protein from degradation, a site of sequestration that regulates or limits telomerase activity, or a site for maturation of the protein. Also the nucleolus might be an assembly machine where telomerase active RNP complex forms. In eukaryotes, ribosomal proteins and rRNAs can be assembled into ribosomal subunits in the nucleolus [32]. We favor the hypothesis that telomerase RNP biogenesis, like the biogenesis of ribosomes and some snRNPs, occurs in

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FIG. 5. Effects of WT-hTERT and mutants on telomerase activities and telomere length. GM847 (A and B) or 293 (C) cells were stably transfected with pCI-neo vector (V) or wild-type hTERT (WT), dominant negative mutated hTERT (DN), N-terminal two-point-mutated hTERT (M2), or N-terminal four-point-mutated hTERT (M4). (A) Telomerase relative activity measured by the Telo TAGGG Telomerase PCR ELISAPLUS kit. (B) Telomerase activity measured by the TRAPEZE Telomerase Detection kit. (C) Telomere length measurement of stably transfected 293 cell lines after 36 passages. A DNA size marker is showed on the left.

the nucleolus, followed by transit of the RNP to the nucleoplasm. Mitchell et al. tried to determine whether the nucleolar population of mature hTR was predominantly associated with an active or inactive telomerase RNP. Unfortunately, they did not succeed because human telomerase activity assays from purified nucleolar fractions are not reliably quantitative [33]. Here we used a different strategy to test this hypothesis. We successfully identified a nucleolar targeting signal at the extreme N-terminus of hTERT protein. We observed that N-terminal point mutations affected the occurrence of protein concentrating in the nucleolus. Then we further investigated the RNP complex formation in stably transfected cells by IP–Northern blot. The result revealed that RNP complex formation was interrupted in M4-hTERT transfected cells although no single arginine was necessary for this function. Recently an in vitro study showed that substitution mutations in this

region affected telomerase activity but did not affect interaction with hTR in vitro [12]. It appears that the disruption of RNP complex formation in M4-hTERT transfected cells is not due to interrupting hTR binding sites in hTERT. To confirm the significance of the RNA binding studies, we measured telomerase activities in the GM847 stably transfected cells. As we mentioned before, GM847 cells are an hTERT-negative/hTR-positive cell line. The advantage of using this cell line is that we can study only the ectopic hTERT activity in this system. The dramatic decrease of the telomerase activity in M4-hTERT-transfected cells indicates that the four point mutations also affected telomerase function. Further, similar localization of GFP tagged hTERT and deletion constructs was seen in SV40 transformed WI38 cells that lack hTR, so that the nucleolar localization or exclusion of hTERT was not mediated by association with hTR. More evidence was obtained by

NUCLEOLAR LOCALIZATION OF hTERT IS CORRELATED WITH FUNCTION OF hTERT

measuring telomere length of stably transfected 293 cells because GM847 cells have an alternative mechanism to maintain their telomere length [34]. Consistent with the telomerase activity assay, telomere length of M4-hTERT transfected 293 cells decreased faster than the control groups after 36 population doublings. All these results support our hypothesis that telomerase active RNP complex was formed in the nucleolus of mammalian cells. We thank Dr. Robert A. Weinberg for providing us with hTERT cDNA plasmids. This work is supported by the Ellison Medical Foundation (Bethesda, MD 20814-5226).

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