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Departments of Pharmacology and Cancer Biology,1 Pathology,2 Radiation Oncology, Duke University Medical Center, Durham, North Carolina 277103
Received 3 December 2001/ Returned for modification 6 January 2002/ Accepted 17 May 2002
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ABSTRACT |
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INTRODUCTION |
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The core of telomerase is composed of a catalytic protein subunit, termed telomerase reverse transcriptase (TERT), that reverse transcribes a template region of an accompanying RNA subunit (TR) onto the ends of telomeres as DNA. The TERT protein is conceptualized as consisting of three regions. The amino terminus (N terminus) is rather basic and contains at least four domains that appear to be evolutionarily conserved. In a number of eukaryotes, the N terminus is essential for telomerase activity, binding to the RNA subunit or multimerization (3, 5-7, 9, 16, 26, 49). The N terminus of human TERT (hTERT) also contains a domain, termed the region that dissociates activities of telomerase (DAT), which is essential for telomere elongation but dispensable for catalytic activity (3). A single mutation in the same region of the yeast catalytic subunit gives rise to a similar phenotype (16), suggesting an evolutionarily conserved function of telomerase aside from catalysis. The central region of TERT from a variety of organisms contains seven evolutionarily conserved motifs that define the catalytic region of reverse transcriptases and in some instances have been shown to be essential for telomerase enzyme activity (33).
The carboxyl terminus (C terminus) is the least-characterized region of TERT. For humans, this region contains a CRM1 and a 14-3-3 binding site, which appear to regulate nuclear export of the protein (42). A region of the C terminus is also required for protein multimerization (2, 6, 7). Alignments of the C-terminal hTERT amino acid sequence with the TERTs of other, more distant organisms show only weak conservation (38). These sequence divergences could underlie differences in the biological function of telomerase in higher versus lower eukaryotes. Indeed, similar although not identical changes to the sequence of the human and yeast catalytic subunits can give rise to different phenotypes. For example, the addition of a hemagglutinin (HA) epitope tag to the C terminus of hTERT has little effect on telomerase in vitro enzyme activity but impedes the ability of the enzyme to elongate telomeres in a number of human cells (13, 21, 36, 54). On the other hand, addition of the same tag to the C terminus of the yeast protein (also containing a deletion of a terminal repeat not found in other TERT molecules) has no detectable effect on its function (15). Different phenotypes also arise from experimental truncations made to the yeast and human TERT proteins. A 
153 mutation that truncates the entire C terminus of yeast TERT (termed Est2) five amino acids downstream from the motif Ei (or RT7) (38) and adds four amino acids before reaching a stop codon does not compromise cell viability (16). In contrast, a deletion of 186 amino acids from the C terminus of hTERT that truncates the protein 10 amino acids downstream of motif E (or RT7) (38) was unable to reconstitute enzyme activity in rabbit reticulocyte lysate (5). Taken together, these results suggest that the C terminus of hTERT may contain domains required for a fully functional enzyme that are not conserved in lower eukaryotes. Identifying such domains represents a key step in elucidating novel functions of the human enzyme.
To search for novel domains of human telomerase involved in the biological function of the enzyme, we took advantage of the fact that ectopic expression of hTERT can immortalize human cells, a recognizable biological phenotype. Indeed, relying on cell death as a measure of telomerase function has been a successful means of characterizing mutants of the catalytic subunit of the enzyme in yeast (15, 16, 34, 49) and humans (3). Capitalizing on this approach, we screened a panel of 41 tandem substitution mutants in the C terminus of hTERT for biological and biochemical activity. Specifically, each mutant of hTERT was stably introduced into transformed human cells prior to crisis. Cell extracts were prepared from the resultant cell lines and assayed for biochemical telomerase catalytic activity in vitro, while the corresponding cultures were monitored for signs of crisis. From this analysis we identified mutations in the C terminus that completely inactivated the enzyme, had no effect on telomerase function, or, most interestingly, resulted in the catalytically active, biologically dysfunctional DAT phenotype. Compiling these mutants, we identified discrete regions required for the biochemical and biological functions of the human telomerase enzyme.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture.
Using standard protocols as previously described (3, 13), HA5 cells and HA1 cells (46) were infected at population doubling (pd) 
45 to 50 with amphotrophic retroviruses derived from the above-described pBabehygro constructs. Stable polyclonal populations were selected in medium supplemented with 100 µg of hygromycin/ml. The first plate to reach confluence under selection was arbitrarily defined as pd 0. All cell lines were cultured until crisis or until the cells had divided at least 20 pds beyond telomerase-negative cells. Crisis was defined as the period when plates failed to become confluent after 20 to 30 days and exhibited massive cell death.
RT-PCR of hTERT mRNA, telomerase activity, and telomere length assays. For reverse transcriptase (RT) PCR analysis of total or endogenous hTERT mRNA or the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, total cellular RNA was prepared with the reagent RNazol (Teltest B, Friendswood, Tex.) according to the manufacturer's instructions, and RT-PCR was carried out with primers specific for the aforementioned transcripts as previously described (20). Reaction products were resolved on 10% polyacrylamide gels, dried, and exposed to a phosphorimager screen.
To detect telomerase activity, cell lysates were prepared in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate buffer (25). Samples were normalized according to protein concentration and were diluted in the same buffer to a concentration of 0.1 µg/ul prior to assaying for activity. One half of the sample was heated at 85°C for 2 min to inactivate telomerase, whereas the other half was directly assayed or serially diluted in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate buffer for dilution experiments. Two microliters of extracts was assayed using a PCR-based telomerase assay, after which the reaction products were resolved on 10% polyacrylamide gels, dried, and exposed to a phosphorimager screen as previously described (25). Activity was calculated as the radioactive counts derived from the experimental extract, minus the heat-treated control, divided by the internal standard as previously described (25). Percent activity of NAAIRS mutants was determined by dividing extract activity by the activity detected in extracts prepared from HA5 cells stably expressing transgenic FLAG-hTERT and multiplying by 100.
Telomeres were visualized by Southern hybridization of HinfI and RsaI restriction enzyme-digested genomic DNA with the 32P-labeled telomeric (C3TA2)3 oligonucleotide exactly as previously described (12), with the exception that 3 µg of digested DNA was resolved and the washes were performed with 10x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Telomere length was determined by analyzing the modal signal via phosphorimager analysis and correlating the peak position with a molecular weight ladder.
Immunoblotting. To assay for FLAG-hTERT protein expression, 293 cells were transiently transfected with the pCMV5 vector or derivatives encoding hTERT, FLAG-hTERT, or FLAG-hTERT NAAIRS mutants using the reagent Lipofectamine Plus in accordance with the manufacturer's protocol (GibcoBRL, Gaithersburg, Md.). Cells were harvested 48 h after transfection and lysed in the presence of 1% Triton X-100, 10 mM Na2HPO4-Na2HPO4 (pH 7.0), 150 mM NaCl, and 5 mM EDTA (pH 8.0), boiled, and then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, Mass.) and immunoblotted with the anti-FLAG M2 (Sigma, St. Louis, Mo.) or the anti-actin I-19 (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) antibody. Blots were washed three times for 10 min in 1x Tris-borate EDTA supplemented with 0.02% Tween 20 at room temperature and then treated with ECL reagent following the manufacturer's protocol (Amersham Pharmacia Biotech, Piscataway, N.J.).
hTR-hTERT and hTERT-hTERT coimmunoprecipitations. 32P-labeled human TR (hTR) and U6 RNAs were produced with the T7 coupled maxiscript system (Ambion, Austin, Tex.) using 1 µg of linearized pBSK-hTR or pBSK-U6. Unincorporated 32P nucleotides were removed by G-25 gel filtration minispin columns (Amersham Pharmacia Biotech). 35S-labeled protein was synthesized in vitro using the T7 quick-coupled TNT system by incubating 1 µg of plasmid pCIneo-FLAG-hTERT-FLAG, pCIneo-FLAG-hTERT-FLAG-NAAIRS +512, +998, +1034, +1088, +1118, or, as a control, pCMV-HDAC in rabbit reticulocyte lysate in the presence or absence of 3 µl of labeled RNA following the manufacturer's instructions (Promega). For experiments showing binding specificity between hTR and U6, 1.5 µl of labeled RNA was used.
Immunoprecipitations were done as described previously (3) using either 9 µg of the anti-FLAG M2 monoclonal antibody (Sigma) prebound to 25 µl of GammaBind G-Sepharose (Amersham Pharmacia Biotech) or 10 µl of anti-FLAG M2 affinity gel. In some cases, 0.5 M urea was added to all final washes. The immunoprecipitate was then resolved by SDS-PAGE. Gels were dried and then exposed to film. When required, a blocking screen was placed between the gel and film to differentiate 32P from 35S signals. Immunoprecipitation of multimeric hTERT complexes was performed as previously described (3). FLAG-tagged proteins and glutathione S-transferase (GST)-tagged proteins were transcribed and translated separately in the presence of either 4 µl of [35S]methionine or 0.5 µl of [35S]methionine and 1 µl of cold methionine, respectively, in the TNT rabbit reticulocyte lysate system (Promega). The plasmids used for the TNT reaction included those mentioned above, as well as pCIneo-FLAG-hTERT-FLAG-NAA +1127 and pCIneo-GST-hTERT-NAAIRS +1127. Reaction mixtures were incubated for 40 min at 30°C, mixed with the appropriate reactions, and then incubated for an additional 60 min at 30°C. Complexes were then immunoprecipitated as described above.
Cytogenetics. Cells were arrested in metaphase using 10 ng of Colcemid (Gibco Life Technologies, Long Island, N.Y.)/ml for 90 min. Cells were then incubated in hypotonic solution (0.75 M KCl) and fixed in methanol-acetic acid (3:1). Slides were prepared and stained using G-banding. Metaphase spreads were scored for telomere fusions irrespective of ploidy.
YFP localization. To assay for YFP-hTERT protein localization, the human osteosarcoma cell line U2OS was grown on poly-D-lysine (molecular mass, >300,000 kDa; Sigma)-coated coverslips. Cells were then transiently transfected with the pEYFP-C1 vector or derivatives encoding YFP-FLAG-hTERT, YFP-FLAG-hTERT NAAIRS1034, YFP-FLAG-hTERT NAAIRS1127 using the reagent Fugene-6 (Roche, Indianapolis, Ind.) in accordance with the manufacturer's protocol. Forty-eight hours later, cells were observed under phosphate-buffered saline at x400 magnification.
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RESULTS |
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-helices and ß-sheets (48) and is thus useful for mapping domains since it disrupts primary amino acid sequence while being less likely to distort the overall structure of the protein (3, 43). In this manner, a large region of hTERT could be mutated with a manageable number of genetic manipulations. In total, 41 tandem substitution mutations in the C terminus of hTERT were created by site-directed mutagenesis, starting at amino acid +890 and ending at the stop codon (Fig. 1A).
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Class one: mutations that inactivate biochemical and biological function of hTERT. To determine the effect of the mutations on biochemical activity, cellular extracts were isolated from all 41 cell lines as well as the two control lines and assayed for in vitro telomerase enzyme activity. Lysates were incubated with a primer that can be elongated by telomerase, and then the reaction products were PCR amplified and resolved to visualize the 6-bp ladder indicative of telomerase activity (25). As a control, duplicate extracts were heat treated to inactivate telomerase prior to assaying for enzyme activity. We found that the mutants displayed different levels of telomerase activity. For example, by dilution of cell lysates, we showed that extracts from HA5 cells infected with FLAG-hTERT or the representative +1022 mutant have similar levels of activity, as characterized by the heat-sensitive 6-bp ladder. Significantly lower levels of telomerase activity were measured in other mutants. For example, telomerase activity was detected in the representative +920 mutant at a protein concentration of 0.03 µg/ul, but barely at a protein concentration of 0.01 µg/ul, corresponding to an approximate fivefold decrease in activity compared to wild-type FLAG-hTERT-infected cells. Enzyme activity was extremely weak in NAAIRS mutants such as the +1118 mutant and undetectable in the representative +1088 mutant, even at the highest protein concentration tested (Fig. 1B).
We therefore classified the mutants as reconstituting telomerase in vitro enzyme activity to either ++ levels (high), + levels (medium), +/- levels (low), or -levels (undetectable), based on the signal intensity of telomerase products (Table 1). Combining these data, a pattern emerged for the regions of the C terminus of hTERT essential for telomerase catalytic activity (Fig. 1A). In total, four novel regions, termed essential region I (E-I), E-II, E-III, and E-IV, which encompass most of the C terminus, were found to be required for easily detectable (++ or + levels) in vitro telomerase activity. Consistent with the boundaries of these regions, alignment of the mouse, human, and Arabidopsis TERT protein sequences demonstrate that these areas tend to be more conserved. This type of mutagenesis also successfully pinpointed the RT motifs 6 (or D) and 7 (or E) (22, 24, 28, 34), which are conserved among all reverse transcriptases (39, 50), as being essential for telomerase enzyme activity whereas mutations to the more divergent linker regions were tolerated (Fig. 2). Thus, it is likely that the E regions identified in hTERT represent bona fide regions of the protein required for enzyme function.
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5 kbp at pd 17 (Fig. 3A), which gradually shortened, and then telomeres maintained a stable length of 3 kbp (not shown), as observed in other cell types (37, 51, 54). These cells were monitored in tissue culture for at least 75 pd beyond crisis, corresponding to more than four times the number of cell divisions as with vector control cells (Fig. 3B). Conversely, the telomere length of the four cell lines expressing the mutated versions of FLAG-hTERT had already shortened to a length of 3 kbp by pd 8 to 13 (Fig. 3A). Accordingly, these mutants failed to proliferate more than the negative-control cells, entered crisis, and died (Fig. 3B). Indeed, all telomerase-negative cell lines were mortal (Table 1), indicating that the mutations that greatly reduced or abolished telomerase activity failed to immortalize the human cells.
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The absence of telomerase enzyme activity in FLAG-hTERT proteins containing NAAIRS mutations in regions E-I through E-IV could potentially have been due to a decrease in protein stability, as observed with some mutants of yeast TERT that display the same phenotype (16). We have been unable to detect ectopic hTERT in cells stably infected with the described hTERT retrovirus by immunoblotting either with an antibody specific for a FLAG tag introduced into the N terminus of hTERT or with an anti-hTERT antibody (not shown). However, FLAG-hTERT can be detected by immunoblot analysis when transiently expressed in 293 cells (3, 7, 30). Expression of FLAG-hTERT in 293 cells should identify any gross defects affecting protein stability. Therefore, epitope-tagged NAAIRS mutants and controls (empty vector or FLAG-tagged or untagged versions of wild-type hTERT) were transiently overexpressed in 293 cells. A protein of 130 kDa, corresponding to the size of hTERT (8, 14, 35, 47), was detected by Western blot using an anti-FLAG antibody in cells transfected with the FLAG-hTERT but not the hTERT construct (Fig. 3C). In the case of the representative mutants in regions E-I through E-IV (+998, +1034, +1088, and +1118), the NAAIRS FLAG-hTERT proteins were detected at levels reflecting that observed with the wild-type FLAG-hTERT control (Fig. 3C). Similarly, other mutations in these regions did not abrogate protein expression (Table 1). These data suggest that the total, or near total, loss of telomerase activity upon mutation of region E-I, E-II, E-III, or E-IV was unlikely to be related to a drastic loss of protein expression. Thus, the majority of the C terminus of hTERT contains regions that are essential for enzyme activity, as evidenced by substitutions that inactivate the known functions of telomerase.
Expression of hTERT and hTR in an environment that facilitates complex formation is sufficient to reconstitute a catalytically active telomerase enzyme in vitro (4, 8, 23, 47). This implies that the loss of in vitro telomerase catalytic activity observed in cells expressing the E-region mutants of hTERT could be attributed to either a defect in the interaction with hTR or some other intrinsic defect in catalysis. To differentiate between these possibilities, we assayed the association of hTR with wild-type and mutant hTERT. 35S-labeled recombinant wild-type FLAG-hTERT-FLAG protein was generated in rabbit reticulocyte lysates in the presence of 32P-labeled in vitro-transcribed hTR RNA. The FLAG-hTERT-FLAG protein was then immunoprecipitated with an anti-FLAG antibody and resolved by SDS-PAGE. In agreement with others (4, 7), we observe that the hTR RNA is coimmunoprecipitated with the recombinant FLAG-hTERT-FLAG protein (Fig. 4A). The specificity of this interaction was demonstrated by four experiments. First, hTR did not associate with an irrelevant FLAG-tagged protein (HDAC), despite the fact that this protein was readily produced and immunoprecipitated with the anti-FLAG antibody (Fig. 4A). Second, we show that the proteins are specifically immunoprecipitated by their FLAG tag, as the HA epitope-tagged version of hTERT was not immunoprecipitated by the anti-FLAG antibody (Fig. 4B). Third, the +512 mutation in one of the known RNA-binding regions of hTERT (3, 26, 32) greatly diminished its in vitro association with hTR (Fig. 4C). Fourth, an irrelevant RNA (U6) showed only negligible binding to both wild-type hTERT and the hTR-binding mutant (Fig. 4C).
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Class two: mutations that do not affect the biochemical and biological function of hTERT. Between regions E-I to E-IV were portions of the protein that were poorly conserved and where NAAIRS substitutions had little or no measurable effect on enzyme activity (Fig. 1A). Correspondingly, mutations to these regions did not diminish the ability of the hTERT protein to immortalize HA5 cells (Table 1). The presence of telomerase activity in these cells was ascribed to the ectopic expression of FLAG-hTERT. First, we sequenced the cDNA derived from the ectopic expressed transcripts and confirmed that only the mutant TERT was present in the cells (not shown). Second, we amplified by RT-PCR RNA isolated at late passage from all nine cell lines that comprised this class of mutants with primers specific for total hTERT mRNA or for the endogenous hTERT transcript. Although a strong signal was detected in all cell lines by using primers for total hTERT mRNA, the endogenous hTERT mRNA could not be detected in these cell lines despite the fact that this transcript was readily amplified in an hTERT-expressing control cancer cell line (Fig. 5 and data not shown). The absence of endogenous hTERT RNA was not due to a limitation of RT-PCR, since we could dilute RNA from telomerase-positive cells 64-fold and still readily detect the transcript (not shown). Thus, the activity detected in cells infected with this class of mutants is derived from the ectopically expressed mutant protein and not activation of the endogenous gene. The regions which tolerate substitutions may therefore act as linkers between the essential regions.
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14 (Fig. 6A). This inability of the +1127 mutant to immortalize cells was attributed to a telomere defect. Similar to vector control-infected cells, cells expressing the +1127 mutant exhibited shortened telomeres of approximately 2.5 kb after nine pds (Fig. 6B). This was in marked contrast to cells expressing wild-type hTERT, which displayed much longer telomeres of 6 kb after the same number of pds. Furthermore, in addition to telomere shortening, cells expressing the +1127 mutant demonstrated a high frequency of telomere fusion events. Specifically, the cells expressing the +1127 mutant had an eightfold increase in dicentric chromosomes (Fig. 6C), compared to the same cells expressing wild-type hTERT (83 versus 11 fusion events per 50 metaphase spreads).
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140 pds beyond crisis) that activate endogenous hTERT (Fig. 6E). Thus, the catalytic activity of the +1127 mutant was at a level sufficient for cellular immortalization. This catalytically active but biologically dead phenotype is analogous to that displayed by mutations to the DAT domain in the N terminus of the protein (3); hence we have named this novel C-terminal region of the protein C-DAT. To more precisely identify the amino acids defining the C-DAT region, we divided the NAAIRS sequence in two, substituting the sequence F1127KT with NAA or replacing the sequence I1130LD with IRS. When these two mutants were introduced into HA5 cells, both restored in vitro telomerase activity (Fig. 7A) but only the F1127KT to NAA substitution recapitulated the phenotype of the +1127 mutant, failing to extend the life span of the cells (Fig. 7B) while only moderately decreasing telomerase activity. We note that not all amino acid substitutions in the F1127KT region led to a DAT phenotype. For example, the +1124 mutant that more conservatively substituted these amino acids did not affect enzyme function (Fig. 7B); similarly single alanine substitutions also did not have any effect on in vitro or in vivo telomerase function (Fig. 7). We therefore define the three-amino-acid-sequence F1127KT as the C-DAT region, being essential for the ability of the enzyme to elongate telomeres in vivo.
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DISCUSSION |
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The four regions denoted E-I, -II, -III, and -IV, identified as being essential for enzyme activity, were separated by spacer regions in which substitutions were less deleterious to telomerase activity. Four lines of reasoning suggest that NAAIRS substitution to these E regions disrupted some precise function of the enzyme, as opposed to globally altering protein folding. First, substitution with the sequence NAAIRS should be rather nonintrusive since the number of amino acids is unchanged and because the sequence NAAIRS can presumably conform to different secondary structures (48). Second, this type of mutagenesis had been used successfully to finely map the binding site of E2F in the pocket domain of Rb (43), RNA binding, and other domains in the N terminus of hTERT (3) and now to identify known RT motifs in hTERT. Third, similar levels of protein were detected in 293 cells expressing the various mutant forms of hTERT, arguing that the mutant proteins were not grossly misfolded and targeted for degradation, although this analysis may not detect more subtle defects when the proteins are expressed in the HA5 cells. Fourth, the association of hTR RNA was not disrupted through NAAIRS substitutions in regions E-I to E-IV.
In an alignment of the human, mouse, and Arabidopsis TERT protein sequences, the four essential regions were found to reside in the most conserved regions of the C terminus (Fig. 1A). Region E-I and RT motif 7 (or E) form one continuous stretch of sequence essential for enzyme activity. In yeast, RT motif 7 (or E) and the downstream C-terminal extension have been implicated in processive nucleotide addition during synthesis of a single telomeric repeat (38). While the present study also suggests that the function of region E-I may be related to RT motif 7 (or E), this particular defect in nucleotide addition was not noted. In addition, a highly conserved threonine residue at position +938 lies in region E-I beyond the defined RT regions, once again suggesting that E-I function may in part be related to the conserved RT motif 7 (or E). The E-I region is also implicated in protein localization and contains a putative NES that has been reported to interact with the protein CRM1 in vitro (42). Region E-II encompasses the reported binding site of 14-3-3 (42), suggesting that the proposed amphipathic helix in this region is essential not only for binding to this class of proteins but also for enzyme activity. The function of the remaining E regions remains to be elucidated. However, mutations to the E-regions did not affect hTR binding, nor did they affect protein stability, suggesting that these regions are important for some step in enzyme catalysis.
Perhaps most interesting is the observation that the +1127 NAAIRS mutation defining the C-DAT region completely abolished the biological function of telomerase while only partially affecting in vitro enzyme activity (Fig. 6). Since the level of enzyme activity was slightly lower than that of mutants that allowed cellular immortality, we investigated whether the inability to maintain telomeres and immortalize cells was related to low catalytic activity or some other yet-unidentified biological activity of hTERT. We compared the activity of the C-DAT mutant in precrisis HA1 cells to endogenous activity of the same cells that had spontaneously immortalized through hTERT expression. Since the telomerase activity of the +1127 C-DAT mutant cell line was comparable to that of the immortal HA1 cells, it was surmised that the C-DAT region was responsible for a crucial telomerase function distinct from catalysis.
Indeed, the phenotype of mutations in this region is identical to that of cells expressing a C-terminal HA-tagged hTERT protein (13, 21, 36, 54). More precise mapping revealed that the mutation of F1127KT to NAA was responsible for the loss of biological function observed in the C-DAT mutant. Since this sequence is just three amino acids away from the position of the HA tag in the hTERT-HA protein, we propose that the sequence F1127KT represents the region that the HA tag exerts its effect upon. Although the exact function of the C-DAT region remains to be resolved, it is apparently unrelated to the ability of the protein to localize to the nucleus or form multimeric complexes. We therefore speculate that the extreme C terminus of hTERT may have evolved to augment some aspect of in vivo telomere elongation, such as binding to telomeres, recognizing the telomere end, or possibly another step in the telomere elongation process itself. We note that the loss of yeast proteins that reside in the telomerase pathway also give rise to telomerase-positive cells which lose telomeric DNA (27). Although speculative, it is possible that the C-terminal region of human TERT defined by mutations to F1127KT may be involved in interactions with mammalian equivalents to these yeast proteins. We also note that the HA tag perturbs binding of hTERT to hnRNP C1 and C2, which are known to colocalize with telomere binding proteins (15a).
While the telomerase-mediated replication of chromosome ends has been a highly conserved theme throughout evolution, the enzyme itself does display differences in its function. For example, the ciliate telomerases are extraordinarily processive (17, 18) and, interestingly, contain specialized motifs not conserved in other organisms (10, 29). We now define several essential regions required for the full biological function of the human enzyme that apparently are not conserved in lower eukaryotes. The human telomerase enzyme does differ, for example, from yeast telomerase in its regulation, fidelity, and association with a much smaller RNA subunit (11, 45). In addition, the telomere repeat sequence, length, and perhaps even configuration differ between these two organisms (19, 52). We speculate that the newly identified domains may encode functions that are unique to higher eukaryotes, which ultimately may be manifested in the replication of telomeres. Since telomerase is activated in the vast majority of human tumors (44), defining such functional domains of the hTERT protein will be important for elucidating how this enzyme functions in malignant cells. Ultimately this may provide insights for the specific inhibition of this pathway for the treatment of human cancers.
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ACKNOWLEDGMENTS |
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S.S.R.B. holds a predoctoral fellowship from the U.S. Department of Defense, and C.M.C. is a Kimmel Scholar. This work was supported by grants from the National Cancer Institute (CA82481) and the V-Foundation.
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FOOTNOTES |
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REFERENCES |
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|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2. Arai, K., K. Masutomi, S. Khurts, S. Kaneko, K. Kobayashi, and S. Murakami. 2002. Two independent regions of human telomerase reverse transcriptase are important for its oligomerization and telomerase activity. J. Biol. Chem. 277:8538-8544.
3. Armbruster, B. N., S. S. Banik, C. Guo, A. C. Smith, and C. M. Counter. 2001. N-terminal domains of the human telomerase catalytic subunit required for enzyme activity in vivo. Mol. Cell. Biol. 21:7775-7786.
4. Bachand, F., and C. Autexier. 1999. Functional reconstitution of human telomerase expressed in Saccharomyces cerevisiae. J. Biol. Chem. 274:38027-38031.
5. Bachand, F., and C. Autexier. 2001. Functional regions of human telomerase reverse transcriptase and human telomerase RNA required for telomerase activity and RNA-protein interactions. Mol. Cell. Biol. 21:1888-1897.
6. Beattie, T. L., W. Zhou, M. O. Robinson, and L. Harrington. 2001. Functional multimerization of the human telomerase reverse transcriptase. Mol. Cell. Biol. 21:6151-6160.
7. Beattie, T. L., W. Zhou, M. O. Robinson, and L. Harrington. 2000. Polymerization defects within human telomerase are distinct from telomerase RNA and TEP1 binding. Mol. Biol. Cell 11:3329-3340.
8. Beattie, T. L., W. Zhou, M. O. Robinson, and L. Harrington. 1998. Reconstitution of human telomerase activity in vitro. Curr. Biol. 8:177-180.[CrossRef][Medline]
9. Bryan, T. M., K. J. Goodrich, and T. R. Cech. 2000. Telomerase RNA bound by protein motifs specific to telomerase reverse transcriptase. Mol. Cell 6:493-499.[CrossRef][Medline]
10. Bryan, T. M., J. M. Sperger, K. B. Chapman, and T. R. Cech. 1998. Telomerase reverse transcriptase genes identified in Tetrahymena thermophila and Oxytricha trifallax. Proc. Natl. Acad. Sci. USA 95:8479-8484.
11. Cohn, M., and E. H. Blackburn. 1995. Telomerase in yeast. Science 269:396-400.
12. Counter, C. M., A. A. Avilion, C. E. Le Feuvre, N. G. Stewart, C. W. Greider, C. B. Harley, and S. Bacchetti. 1992. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J. 11:1921-1929.[Medline]
13. Counter, C. M., W. C. Hahn, W. Wei, S. D. Caddle, R. L. Beijersbergen, P. M. Lansdorp, J. M. Sedivy, and R. A. Weinberg. 1998. Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization. Proc. Natl. Acad. Sci. USA 95:14723-14728.
14. Counter, C. M., M. Meyerson, E. N. Eaton, L. W. Ellisen, S. D. Caddle, D. A. Haber, and R. A. Weinberg. 1998. Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase. Oncogene 16:1217-1222.[CrossRef][Medline]
15. Counter, C. M., M. Meyerson, E. N. Eaton, and R. A. Weinberg. 1997. The catalytic subunit of yeast telomerase. Proc. Natl. Acad. Sci. USA 94:9202-9207.
15. Ford, L. P., J. M. Suh, W. E. Wright, and J. W. Shay. 2000. Heterogeneous nuclear ribonucleoproteins C1 and C2 associate with the RNA component of human telomerase. Mol. Cell. Biol. 20:9084-9091.
16. Friedman, K. L., and T. R. Cech. 1999. Essential functions of amino-terminal domains in the yeast telomerase catalytic subunit revealed by selection for viable mutants. Genes Dev. 13:2863-2874.
17. Greider, C. W. 1991. Telomerase is processive. Mol. Cell. Biol. 11:4572-4580.
18. Greider, C. W., and E. H. Blackburn. 1985. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43:405-413.[CrossRef][Medline]
19. Griffith, J. D., L. Comeau, S. Rosenfield, R. M. Stansel, A. Bianchi, H. Moss, and T. de Lange. 1999. Mammalian telomeres end in a large duplex loop. Cell 97:503-514.[CrossRef][Medline]
20. Hahn, W. C., C. M. Counter, A. S. Lundberg, R. L. Beijersbergen, M. W. Brooks, and R. A. Weinberg. 1999. Creation of human tumour cells with defined genetic elements. Nature 400:464-468.[CrossRef][Medline]
21. Halvorsen, T. L., G. Leibowitz, and F. Levine. 1999. Telomerase activity is sufficient to allow transformed cells to escape from crisis. Mol. Cell. Biol. 19:1864-1870.
22. Harrington, L., W. Zhou, T. McPhail, R. Oulton, D. S. Yeung, V. Mar, M. B. Bass, and M. O. Robinson. 1997. Human telomerase contains evolutionarily conserved catalytic and structural subunits. Genes Dev. 11:3109-3115.
23. Holt, S. E., 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, and M. A. White. 1999. Functional requirement of p23 and Hsp90 in telomerase complexes. Genes Dev. 13:817-826.
24. Kilian, A., D. D. L. Bowtell, H. E. Abud, G. R. Hime, D. J. Venter, P. K. Keese, E. L. Duncan, R. R. Reddel, and R. A. Jefferson. 1997. Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types. Hum. Mol. Genet. 6:2011-2019.
25. Kim, N. W., and F. Wu. 1997. Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP). Nucleic Acids Res. 25:2595-2597.
26. Lai, C. K., J. R. Mitchell, and K. Collins. 2001. RNA binding domain of telomerase reverse transcriptase. Mol. Cell. Biol 21:990-1000.
27. Lingner, J., T. R. Cech, T. R. Hughes, and V. Lundblad. 1997. Three Ever Shorter Telomere (EST) genes are dispensable for in vitro yeast telomerase activity. Proc. Natl. Acad. Sci. USA 94:11190-11195.
28. Meyerson, M., C. M. Counter, E. N. Eaton, L. W. Ellisen, P. Steiner, S. D. Caddle, L. Ziaugra, R. L. Beijersbergen, M. J. Davidoff, Q. Liu, S. Bacchetti, D. A. Haber, and R. A. Weinberg. 1997. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 90:785-795.[CrossRef][Medline]
29. Miller, M. C., J. K. Liu, and K. Collins. 2000. Template definition by Tetrahymena telomerase reverse transcriptase. EMBO J. 19:4412-4422.[CrossRef][Medline]
30. Mitchell, J. R., E. Wood, and K. Collins. 1999. A telomerase component is defective in the human disease dyskeratosis congenita. Nature 402:551-555.[CrossRef][Medline]
31. Morgenstern, J. P., and H. Land. 1990. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18:3587-3596.
32. Moriarty, T. J., S. Huard, S. Dupuis, and C. Autexier. 2002. Functional multimerization of human telomerase requires an RNA interaction domain in the N terminus of the catalytic subunit. Mol. Cell. Biol. 22:1253-1265.
33. Nakamura, T. M., and T. R. Cech. 1998. Reversing time: origin of telomerase. Cell 92:587-590.[Medline]
34. Nakamura, T. M., G. B. Morin, K. B. Chapman, S. L. Weinrich, W. H. Andrews, J. Lingner, C. B. Harley, and T. R. Cech. 1997. Telomerase catalytic subunit homologs from fission yeast and human. Science 277:955-959.
35. Nakayama, J., H. Tahara, E. Tahara, M. Saito, K. Ito, H. Nakamura, T. Nakanishi, T. Ide, and F. Ishikawa. 1998. Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nat. Genet. 18:65-68.[CrossRef][Medline]
36. Ouellette, M. M., D. L. Aisner, I. Savre-Train, W. E. Wright, and J. W. Shay. 1999. Telomerase activity does not always imply telomere maintenance. Biochem. Biophys. Res. Commun. 254:795-803.[CrossRef][Medline]
37. Ouellette, M. M., M. Liao, B. S. Herbert, M. Johnson, S. E. Holt, H. S. Liss, J. W. Shay, and W. E. Wright. 2000. Subsenescent telomere lengths in fibroblasts immortalized by limiting amounts of telomerase. J. Biol. Chem. 275:10072-10076.
38. Peng, Y., I. S. Mian, and N. F. Lue. 2001. Analysis of telomerase processivity: mechanistic similarity to HIV-1 reverse transcriptase and role in telomere maintenance. Mol. Cell 7:1201-1211.[CrossRef][Medline]
39. Poch, O., I. Sauvaget, M. Delarue, and N. Tordo. 1989. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 8:3867-3874.[Medline]
40. Sankar, S., and A. G. Porter. 1992. Point mutations which drastically affect the polymerization activity of encephalomyocarditis virus RNA-dependent RNA polymerase correspond to the active site of Escherichia coli DNA polymerase I. J. Biol. Chem. 267:10168-10176.
41. Sedivy, J. M. 1998. Can ends justify the means?: telomeres and the mechanisms of replicative senescence and immortalization in mammalian cells. Proc. Natl. Acad. Sci. USA 95:9078-9081.
42. Seimiya, H., H. Sawada, Y. Muramatsu, M. Shimizu, K. Ohko, K. Yamane, and K. Tsuruo. 2000. Involvement of 14-3-3 proteins in nuclear localization of telomerase. EMBO J. 19:2652-2661.[CrossRef][Medline]
43. Sellers, W. R., B. G. Novitch, S. Miyake, A. Heith, G. A. Otterson, F. J. Kaye, A. B. Lassar, and W. G. Kaelin, Jr. 1998. Stable binding to E2F is not required for the retinoblastoma protein to activate transcription, promote differentiation, and suppress tumor cell growth. Genes Dev. 12:95-106.
44. Shay, J. W., and S. Bacchetti. 1997. A survey of telomerase activity in human cancer. Eur. J. Cancer 33:787-791.
45. Singer, M. S., and D. E. Gottschling. 1994. TLC1: template RNA component of Saccharomyces cerevisiae telomerase. Science 266:404-409.
46. Stewart, N., and S. Bacchetti. 1991. Expression of SV40 large T antigen, but not small t antigen, is required for the induction of chromosomal aberrations in transformed human cells. Virology 180:49-57.[CrossRef][Medline]
47. Weinrich, S. L., R. Pruzan, L. Ma, M. Ouellette, V. M. Tesmer, S. E. Holt, A. G. Bodnar, S. Lichtsteiner, N. W. Kim, J. B. Trager, R. D. Taylor, R. Carlos, W. H. Andrews, W. E. Wright, J. W. Shay, C. B. Harley, and G. B. Morin. 1997. Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat. Genet. 17:498-503.[CrossRef][Medline]
48. Wilson, I. A., D. H. Haft, E. D. Getzoff, J. A. Tainer, R. A. Lerner, and S. Brenner. 1985. Identical short peptide sequences in unrelated proteins can have different conformations: a testing ground for theories of immune recognition. Proc. Natl. Acad. Sci. USA 82:5255-5259.
49. Xia, J., Y. Peng, I. S. Mian, and N. F. Lue. 2000. Identification of functionally important domains in the N-terminal region of telomerase reverse transcriptase. Mol. Cell. Biol. 20:5196-5207.
50. Xiong, Y., and T. H. Eickbush. 1990. Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J. 9:3353-3362.[Medline]
51. Yang, J., E. Chang, A. M. Cherry, C. D. Bangs, Y. Oei, A. Bodnar, A. Bronstein, C. P. Chiu, and G. S. Herron. 1999. Human endothelial cell life extension by telomerase expression. J. Biol. Chem. 274:26141-26148.
52. Zakian, V. A. 1996. Structure, function, and replication of Saccharomyces cerevisiae telomeres. Annu. Rev. Genet. 30:141-172.[CrossRef][Medline]
53. Zakian, V. A. 1995. Telomeres: beginning to understand the end. Science 270:1601-1607.
54. Zhu, J., H. Wang, J. M. Bishop, and E. H. Blackburn. 1999. Telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening. Proc. Natl. Acad. Sci. USA 96:3723-3728.
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), II (
), III (
), and IV (
). (C) Mutations in regions E-I, -II, -III, and -IV do not alter RNA or protein levels of hTERT. Total RNA prepared from the same aforementioned cell lines, or the control HA5 cells stably expressing untagged hTERT, was RT-PCR amplified with primers specific for hTERT or GAPDH. Protein lysates prepared from 293 cells transiently transfected with an empty (vector) mammalian expression vector or one encoding FLAG-hTERT, hTERT, or representative FLAG-hTERT NAAIRS mutants for each of the four essential regions were resolved by SDS-PAGE and Western blotted with an anti-FLAG or antiactin antibody.
