mTERT cannot substitute for hTERT in immortalization of human fibroblasts.
To compare the activities of mTERT and hTERT, we assessed their ability to prevent the critical telomere shortening that is characteristic of human cells in crisis. A subset of telomeres becomes uncapped during crisis, resulting in chromosomal end-to-end fusions and apoptosis. We therefore studied human BJ fibroblasts that were stably expressing the SV40 early region, which abrogates the replicative senescence response, allowing telomeres to become critically short at crisis with continued cell division. The expression of TERT alleles in this cellular context is therefore a test of their ability to synthesize and stabilize the shortest telomeres in the population. Enforced expression of hTERT reconstituted telomerase activity as measured by the TRAP (Fig. ). Telomerase activity was detected neither in untransduced BJT cells nor in BJT cells that were infected with the retroviral vector alone (Fig. ). The transduction of BJT cells with mTERT retrovirus reproducibly resulted in telomerase activity by TRAP, although the levels of TRAP activity were consistently lower than that in hTERT-transduced cells (Fig. and data not shown). These data suggest that mTERT can assemble with human TERC (hTERC) to allow the formation of enzymatically active telomerase ribonucleoprotein complexes. To assess directly the ability of mTERT to interact with hTERC, coimmunoprecipitation experiments were performed in 293T cells that were transfected with expression plasmids for hTERC and either FLAG-tagged hTERT or FLAG-tagged mTERT. TERT protein in lysates from transfected cells was immunoprecipitated by using anti-FLAG antibody agarose. FLAG antibody efficiently pulled down the tagged TERT proteins. Northern blotting on the affinity-purified complexes showed that hTERC was associated with both hTERT and mTERT at comparable levels (Fig. ). Together, these data show that mTERT efficiently interacts with hTERC in human cells, giving rise to an enzymatically active chimeric telomerase RNP, consistent with previous findings (15
FIG. 1. mTERT cannot immortalize primary human fibroblasts. (A) Expression of mTERT in primary BJT cells reconstitutes telomerase activity by TRAP assay. Note the telomerase elongation products in cells expressing hTERT and mTERT but not with empty vector alone. (more ...)
To determine whether reconstitution of telomerase with mTERT is sufficient to immortalize human fibroblasts, the proliferative capacity of mTERT-BJT cells was compared to that of hTERT-BJT cells through serial passage. Cells were counted and seeded every 3.5 days for up to 60 population doublings (80 days). Cells that were transduced with the empty retroviral vector entered crisis after approximately 10 population doublings, whereas hTERT-BJT cells showed a dramatically increased lifespan, proliferating in an unimpeded fashion for at least 60 population doublings (Fig. ). Despite the efficient reconstitution of telomerase enzymatic activity, mTERT did not extend the replicative lifespan of BJT cells (Fig. ). Both vector-BJT and mTERT-BJT cultures showed characteristic apoptotic morphology (data not shown) as well as elevated rates of chromosomal end-to-end fusions (Table ). In contrast, metaphase chromosomes from hTERT-transduced cells showed no chromosomal end-to-end fusions, indicating that telomeres remain protected in the hTERT cultures (P was 0.001 for hTERT versus mTERT by t test) (Table ). Experiments with BJ fibroblasts and IMR90 fibroblasts lacking expression of the SV40 early region similarly showed that mTERT cannot avert replicative senescence of primary human cells (data not shown). These results show that, unlike hTERT, mTERT cannot prevent chromosomal end-to-end fusions or extend the proliferative lifespan of human cells.
Telomere analysis of stably transduced BJT cellsa
The appearance of end-to-end fusions in mTERT cultures suggests that, despite efficient reconstitution of telomerase enzymatic activity, mTERT cannot synthesize telomere repeats in human cells. To determine whether mTERT can extend human telomeres, telomere lengths were measured by Southern blot analysis. Telomeres increased in length in BJT cells that were transduced with hTERT with advancing cell divisions, while cells that were transduced with vector alone showed telomere shortening (Fig. ). Telomeres in mTERT-BJT cells shortened progressively and were indistinguishable from vector-only controls, with mean telomere lengths at crisis of 5.8 kb and 5.7 kb, respectively (Fig. and Table ). Together, these data indicate that despite strong sequence similarity to hTERT and reconstitution of enzymatically active telomerase complexes in human cells, mTERT cannot effectively extend human telomeres and fails to immortalize human fibroblasts.
The N terminus of hTERT cannot confer upon mTERT the ability to immortalize human cells.
The observation that mTERT reconstitutes enzymatic activity but cannot extend the lifespan of human cells creates a unique opportunity to understand the protein domains and sequences that account for this important difference in function. The similarity between the two proteins allows for the exchange of homologous regions that should conserve the overall structure of TERT while allowing for the investigation of the effect of infrequent but functionally important amino acid differences. To study the effects of these sequence differences on TERT function, we created chimeric TERT proteins by exchanging mouse and human sequences in three main functional domains spanning the TERT open reading frame: (i) the amino-terminal domain containing the essential domain I and TERC-binding domains II and III, (ii) the reverse transcriptase domain, including the T motif and the conserved RT motifs, and (iii) the carboxy-terminal region (Fig. ). Chimeric cDNAs were expressed from retroviruses to characterize their function in primary human cells.
FIG. 2. Analysis of the N-terminal domain and RT domain in immortalization and telomere maintenance. (A) Schematic diagram of TERT delineating the three major domains: N terminus, RT domain, and the C terminus (C-term). Functional regions I, II, III, T, N-DAT, (more ...)
To analyze the importance of sequence differences in the N terminus, this region was exchanged between mouse and human TERT to yield the chimeric proteins HMM and MHH. The nomenclature of these constructs is designed to show the identity, whether mouse or human, of the N terminus, RT domain, and the C terminus, based on position. Stable retroviral expression of the reciprocal mutants HMM and MHH in BJT cells showed that HMM-BJT cells exhibited telomerase activity similar to that of cells expressing wild-type hTERT, whereas telomerase activity was not detected in MHH-BJT cells (Fig. ). The lack of telomerase activity in the MHH mutant was unexpected because mTERT efficiently interacted with hTERC and because the human N-terminal domain could be readily substituted in mTERT to yield an active telomerase complex. To determine whether this mutant demonstrates enzymatic function in vitro, MHH protein was expressed in rabbit reticulocyte lysates in the presence of hTERC RNA. When equal amounts of mTERT and MHH proteins were used to program TRAP reactions, MHH showed a significantly reduced activity compared to that of wild-type mTERT, thus explaining the absence of enzymatic activity in transduced cells (see Fig. S1 in the supplemental material). Although the MHH mutant exhibited reduced enzymatic activity, the HMM mutant was fully active, allowing us to determine whether the human N terminus could confer the ability to immortalize human cells.
To assess the importance of the N-terminal region in immortalization, HMM- and MHH-transduced BJT cells were cultured for approximately 20 population doublings (60 days). Cells that were transduced with empty vector entered crisis by population doubling 13 (day 50) as did MHH-BJT cells, which was expected for an enzymatically impaired mutant (Fig. ). Despite robust enzymatic activity by TRAP, HMM protein did not extend the replicative lifespan of BJT cells. HMM-BJT cells entered crisis by population doubling 18 (day 60) (Fig. ). Metaphase preparations demonstrated chromosomal end-to-end fusions for both MHH-BJT and HMM-BJT (0.47 fusions per metaphase for each mutant) (P was 0.004 for MHH and 0.008 for HMM versus hTERT by t test) (Table ), and both cultures showed apoptotic morphologies (data not shown). Telomere Southern blot analysis demonstrated equivalent telomere shortening for MHH, HMM, and vector-transduced cells (mean telomere lengths at crisis were 5.9 kb and 5.7 kb for HMM and vector, respectively) (Fig. , Table , and data not shown). These data indicate that, despite the strong telomerase activity exhibited by the HMM mutant, the N-terminal domain of human TERT did not allow telomere maintenance or extension of replicative lifespan when combined with the RT and C-terminal domains of mTERT.
The mouse RT domain substitutes effectively for the human RT domain in immortalization and telomere maintenance.
To determine whether the sequence differences that allow immortalization by hTERT but not by mTERT lie in the critical RT domain, mouse and human RT domains were exchanged to yield HMH and MHM proteins. The presence of the mouse RT domain in HMH allowed telomerase activity in BJT cells that was comparable to that of hTERT (Fig. ). Moreover, when expressed in BJT cells through retroviral transduction, the HMH mutant dramatically extended the replicative lifespan for at least 170 population doublings (over 200 days), an activity that was indistinguishable from hTERT (Fig. ). In contrast, empty vector controls and mTERT-transduced cells entered crisis within 15 population doublings (day 50) (Fig. ). Consistent with the ability of the HMH mutant to immortalize cells, chromosomal end-to-end fusions were rare in HMH cultures (0.043 fusions per metaphase) (P was 0.001 for HMH versus mTERT by t test) (Table ). To assess the ability of HMH to maintain telomere length, telomere Southern blot analyses were performed on DNA that was isolated during the serial passage of HMH-BJT cells. Interestingly, telomeres in the bulk population of HMH-BJT cells initially shortened, despite the fact that the HMH mutant prevented telomere uncapping and supported continued cell proliferation (Fig. ). Upon continued cell passage, it became evident that after 90 population doublings, mean telomere length stabilized at approximately 3.9 kb (Fig. and Table ). These data show that the mouse RT domain, in the context of the N-terminal and C-terminal domains from hTERT, is sufficient to maintain telomeres, prevent telomere uncapping, and immortalize BJT cells.
Analysis of the reciprocal mutant MHM revealed an absence of telomerase activity in transduced cells, which was reminiscent of the MHH protein (Fig. ). When expressed in vitro in rabbit reticulocyte lysates with hTERC RNA, MHM also showed a markedly reduced specific activity compared to that of mTERT (see Fig. S1 in the supplemental material). Thus, both the MHH and the MHM mutants exhibit a defect in enzymatic activity, likely specific to the juxtaposition of the mouse N terminus and human RT domain. As anticipated for a mutant with such a functional defect, MHM-transduced BJT cells entered crisis simultaneously with empty vector controls, after 12 population doublings (by day 50) (Fig. ). In addition, chromosomal end-to-end fusions were elevated just as in empty vector controls, consistent with an absence of telomere maintenance in these cultures (data not shown). Together, these data show that amino acid differences in neither the N-terminal domain nor the RT domain explain the inability of mTERT to maintain telomeres and prevent crisis in primary human cells.
The C-terminal domain of TERT controls immortalization and telomere maintenance.
The preceding data demonstrate that the substitution of the N-terminal sequences of hTERT into the mTERT protein does not allow telomere maintenance in human cells and that the mouse RT domain can effectively substitute for the human RT sequences in telomere maintenance and immortalization. These results show that the inability of mTERT to maintain telomeres in human cells is not due to sequence variation within these domains. To determine whether amino acid variation in the C terminus accounts for the differences in telomere maintenance between mTERT and hTERT, we exchanged carboxy-terminal domains between the two orthologues, creating HHM and MMH TERT mutants. Retroviruses expressing HHM and MMH proteins were used to infect BJT cells, and cell protein extracts were collected to analyze telomerase activity. Interestingly, the transfer of the 120-amino-acid C-terminal domain reversed the relative TRAP activity of the mutants such that MMH exhibited significantly higher activity than HHM. The enzymatic activity of HHM was comparable to that of mTERT, whereas the activity of MMH was elevated to that of hTERT (Fig. ).
FIG. 3. C terminus controls enzymatic activity and immortalization. (A) C terminus of hTERT dictates activity level. TRAP analysis of BJT cells that were transduced with retroviruses expressing HHM, MMH, hTERT, and mTERT (IC, internal control). (B) Serial passage (more ...)
To assess the effect of exchanging C-terminal domains on cellular immortalization, the proliferative capacity of MMH- and HHM-transduced BJT cultures was studied through serial passage. Cells expressing HHM showed a finite lifespan that was limited by crisis (population doubling 14, day 60) in a fashion similar to that of mTERT-transduced cells and empty vector controls (Fig. ). These results indicate that the inability of mTERT to immortalize human cells is due to sequence divergence in the C terminus. Indeed, MMH-transduced cells showed a dramatically extended lifespan comparable to that of the cells expressing hTERT. MMH-BJT cultures demonstrated continued proliferation for over 140 population doublings (200 days) (Fig. ). Cytogenetic analysis showed that MMH protein, but not the HHM mutant, efficiently preserved telomere function. MMH-transduced cultures showed only 0.17 chromosomal fusions per metaphase, a rate of fusion similar to that of hTERT (P was 0.003 for MMH versus mTERT by t test). In contrast, cells expressing HHM exhibited a significantly elevated prevalence of chromosomal fusions (0.58 fusions per metaphase) (P was 0.012 for HHM versus hTERT by t test) (Table ). These data show that divergent sequences in the C-terminal domain underlie the differential activity of mouse and human orthologues in immortalization and telomere maintenance of human cells.
To investigate directly the role of the C-terminal domain in telomere length regulation, telomere Southern blot analyses were performed by using genomic DNA that was isolated from cells during serial passage. Mean telomere length decreased progressively in HHM-BJT cells (Fig. ) similarly to that in cells expressing empty vector or wild-type mTERT (Fig. and ). Interestingly, the mean telomere length in the population of HHM-BJT cells at crisis remained long (5.5 kb). In fact, all the cultures that failed to immortalize, including those transfected with HHM, HMM, wild-type mTERT, or empty vector, entered crisis with similar mean telomere lengths (5.5 kb, 5.9 kb, 5.8 kb, and 5.7 kb, respectively). Despite the apparently ample telomere reserve, all these cultures showed similarly elevated rates of chromosomal fusions, which is clear evidence of telomere dysfunction (Table ). In comparison, telomeres shortened in immortally proliferating MMH-BJT cells for as long as 110 population doublings and were then maintained at a stable length of approximately 3.9 kb (Fig. ; Table ). The mean telomere length of proliferating MMH-BJT cells was significantly shorter than that of HHM-BJT cells in crisis, indicating that the MMH mutant may effectively maintain the shortest telomeres in the population.
To investigate whether the MMH mutant immortalizes human fibroblasts by synthesizing telomere repeats on the shortest telomeres, we performed Q-FISH, which allows quantitation of the telomere signal at each chromosome end (69
). In cultures that had been transduced with empty vector and were therefore approaching crisis (pd 25), telomeres showed a broad distribution with a median telomere signal of 827 fluorescence units. In contrast, telomeres were markedly shorter in MMH-BJT cells analyzed after a larger number of population doublings (pd 150), showing a median telomere signal of 359 fluorescence units (P
was <0.0001 by two-tailed Wilcoxon rank sum tests, vector versus MMH) (Fig. ). Although telomeres were, on average, shorter in MMH cells, the number of chromosomal ends with telomere signals below the level of detection was greater in vector-transduced fibroblasts (6.2 signal-free ends [SFEs] per metaphase for vector versus 3.0 SFEs per metaphase for MMH cells, P
was 0.0046). This elevated frequency of SFEs in vector cells is consistent with the observation that vector-transduced cells enter crisis, whereas MMH cells continue proliferating in immortal fashion. Together, these data show that the human C-terminal sequences in MMH allow telomere maintenance at a short set point and that these short telomeres remain capped, as evidenced by the continued proliferation of the culture and absence of chromosomal fusions. In addition to maintaining these short telomeres through telomere synthesis, it is also possible that the presence of telomerase enhances telomere stability in this setting by contributing to the functional cap at chromosome ends (12
FIG. 4. Telomeres are maintained at a short length and within a narrow range in MMH-expressing cells. Q-FISH analysis of MMH-BJT and vector-BJT cells. MMH cells at advanced passage (pd 150) demonstrate a significantly shorter mean telomere length than those for (more ...) An important role for the C1 domain in immortalization.
To understand which residues in the C terminus control immortalization, we compared the sequences in the TERT C-terminal domain among vertebrates, including mice and humans. Sequence alignment shows similarity throughout the length of the C-terminal domain, with blocks of strong conservation separated by smaller regions of divergence (see Fig. S2 in the supplemental material). Based on this analysis, we divided the C terminus into two regions such that divergent residues between the mouse and human orthologues fall equally into each region, C1 or C2 (Fig. ; see Fig. S2 in the supplemental material). Mutants comprising the open reading frame of mTERT with a substitution of the C1 domain from hTERT (mTERT-hC1) and the open reading frame of hTERT with the C1 domain from mTERT (hTERT-mC1) were expressed in BJT cells by retroviral transduction. The substitution of the C1 domain reversed the telomerase activity levels of these mutants; mTERT-hC1 showed significantly increased TRAP activity compared to that of hTERT-mC1 (Fig. ). Thus, the ability of the C-terminal domain to alter telomerase activity, as seen in the HHM and MMH mutants (Fig. ), maps to the C1 domain.
FIG. 5. Human C1 is crucial for extended lifespan and telomere maintenance. (A) TRAP analysis of BJT cells transduced with retroviruses expressing MMH, HHM, mTERT-hC1, hTERT-mC1, mTERT-hC2, hTERT-mC2, hTERT, or mTERT. (B) C1 domain of hTERT is required, but not (more ...)
To analyze the ability of hTERT-mC1 and mTERT-hC1 to extend cellular lifespan, BJT cells that were transduced with retroviruses expressing these alleles were serially passaged for approximately 50 days. Neither hTERT-mC1 nor mTERT-hC1 could extend lifespan, and both cultures entered crisis by population doubling 5 (day 30); this behavior was similar to that of the vector-only control (Fig. ). Consistent with their inability to immortalize BJT cells, both hTERT-mC1- and mTERT-hC1-transduced cells showed an increase in chromosome end-to-end fusions by cytogenetic analysis, with mTERT-hC1-transduced cells demonstrating 1.3 fusions per metaphase (Table and data not shown). These data suggest that the C1 domain harbors important sequences for telomere maintenance and immortalization but that the human C1 sequences are insufficient to allow immortalization in the context of mTERT. The fact that MMH can immortalize cells, whereas mTERT-hC1 cannot, indicates that there are additional residues in the C2 region that are necessary for a fully functional C-terminal domain to facilitate telomere maintenance in human cells.
Exchanging the C2 sequences between mTERT and hTERT further supported a critical role for the C1 domain in immortalization. Substitution of the C2 regions did not alter telomerase activity. Telomerase activity of mTERT-hC2 was similar to that of mTERT, and activity of hTERT-mC2 was unchanged compared to that of hTERT (Fig. ). Interestingly, hTERT-mC2 was capable of immortalizing BJT cells for at least 140 population doublings (200 days), whereas mTERT-hC2-BJT cells entered crisis similarly to controls (less than 5 population doublings, 30 days) (Fig. and data not shown). Consistent with their extended proliferative lifespan, hTERT-mC2-BJT cells showed only 0.075 end-to-end fusions per metaphase, a number similar to that of hTERT-transduced cells in cytogenetic analysis (P was 0.001 for hTERT-mC2 versus mTERT by t test) (Table ). Telomere measurements by Southern blot analysis showed that telomeres shortened progressively in hTERT-mC2-BJT cultures and then stabilized at approximately 4.6 kb (Fig. ; Table ); this was similar to telomere dynamics in immortal HMH-BJT cells (Fig. ) and MMH-BJT cells (Fig. ). The stabilization of telomere length was not due to upregulation of the endogenous hTERT gene because allele-specific RT-PCR showed that endogenous TERT cDNA was undetectable in all retrovirally transduced cultures analyzed (see Fig. S3 in the supplemental material). Taken together, these data show that the sequences in the C terminus that allow immortalization in human cells reside predominantly in the C1 region, although additional residues in the C2 region are likely important in facilitating immortalization in the MMH mutant (Table ).
Summary of activities of TERT mutants
The TERT C terminus controls steady-state protein levels.
To understand the regulation of our TERT mutants at the protein level, each mutant was tagged with an immunoglobulin-binding repeat from SPA, facilitating sensitive and quantitative detection by Western blotting (14
). SPA-tagged TERT proteins were expressed in BJ cells via retroviral transduction, and total cell protein extracts were analyzed by Western blotting. Surprisingly, whereas SPA-hTERT was readily detected by Western blotting, SPA-mTERT protein was expressed at much lower levels (Fig. ). This difference in protein level was also seen with FLAG-hTERT and FLAG-mTERT, indicating that the lower levels of mTERT protein are independent of the specific tag used. In this case, immunoprecipitation with anti-FLAG antibody agarose followed by α-FLAG Western blotting was required to detect FLAG-mTERT protein (Fig. ). To determine whether the difference in steady-state mTERT and hTERT protein levels could be due to variation in mRNA levels, RNA was collected from these cells for Northern blot analysis. Using a probe to the SPA tag sequences, we found no difference in the levels of retroviral transcripts for mTERT and hTERT (Fig. ). Furthermore, indirect immunofluorescence revealed that both hTERT and mTERT proteins were predominantly expressed in the nucleus, therefore the different activities of the TERT orthologues is unlikely to be due to altered compartment of expression (Fig. ). These findings indicate that expressing mTERT and hTERT from the same retroviral promoter results in identical mRNA transcript levels but a pronounced difference in steady-state protein levels.
FIG. 6. hTERT accumulates to markedly higher protein levels than those of mTERT and these differences in protein level are controlled by SS1-2 sequences within the C1 domain. (A) Sequences regulating differences in mTERT and hTERT protein levels localize to the (more ...)
Given this unexpected and marked dissociation between transcript and protein levels, we analyzed each of our tagged TERT mutants in order to map a domain that was controlling TERT protein level. Each SPA-tagged TERT construct was stably expressed in BJ cells via retroviral transduction. SPA-HMM was expressed at low levels, similar to those of SPA-mTERT, indicating that when transferred to mTERT, the N-terminal domain of hTERT does not lead to increased protein levels (Fig. ). SPA-HMH accumulated to levels similar to those of SPA-hTERT, suggesting that sequences in the RT domain are not responsible for the low levels of mTERT protein (Fig. ). To determine whether the remaining domain, the C terminus, dictates protein levels, we analyzed SPA-HHM and SPA-MMH. SPA-HHM protein was expressed at mTERT-like levels, whereas SPA-MMH accumulated to levels similar to those of hTERT (Fig. ). Similar results were obtained in transformed human embryonic kidney cells (293T) and in mouse embryonic fibroblasts, indicating that the difference in protein levels is intrinsic to the mTERT and hTERT proteins and not dependent on cell type (data not shown). These results show that the marked difference in steady-state protein levels between mouse and human TERT orthologues is due to sequence variation in the C-terminal domain.
Sequences within the SS1-2 regions of C1 dictate TERT protein levels.
To further delineate the region in the C terminus of hTERT that controls protein levels, we analyzed SPA-tagged versions of the C1 and C2 region mutants. An exchange of the C1 region between mTERT and hTERT dramatically altered protein levels. Protein levels of hTERT-mC1 were significantly reduced, and conversely, mTERT-hC1 protein levels were markedly increased (Fig. ). In contrast, substitution of the C2 sequences had no effect on protein levels. The levels of neither hTERT-mC2 nor mTERT-hC2 were affected by the exchange of the C2 regions (Fig. ). The results of Northern blot analysis of mRNA transcripts from BJ cells that were stably transfected with each retroviral construct showed that each chimeric cDNA was expressed equally at the mRNA level, supporting the conclusion that the different protein levels are intrinsic properties of each mutant protein (Fig. ). These data demonstrate that sequences in the C1 region control steady-state TERT protein levels in both species.
To delineate the sequences responsible for increased steady-state protein levels, the C1 domain was divided into four regions, denoted SS1 to -4, each approximately 14 amino acids in length (see Fig. S2 in the supplemental material). New chimeric proteins were constructed in which the human steady-state (hSS) sequences were substituted for the endogenous mouse sequences in mTERT, yielding mTERT-hSS2-4, mTERT-hSS3-4, and mTERT-hSS4 chimeric proteins. SPA-tagged mTERT-hSS2-4 demonstrated markedly increased protein levels compared to those of mTERT but slightly reduced levels compared to those of mTERT-hC1, which contains the entire C1 region from hTERT. However, replacing the human SS2 sequence with the mouse SS2 sequence resulted in a dramatic reduction in protein levels, comparable to those of wild-type mTERT (Fig. , mTERT-hSS3-4 and mTERT-hSS4 lanes). Together, these data identify the SS1 and SS2 sequences within the C1 domain as those controlling TERT protein level.
All of the mutations that reduced hTERT protein levels abrogated immortalization, suggesting that these elevated levels are required for telomere maintenance. Interestingly, both the mTERT-hC1 mutant and the mTERT-hSS2-4 mutant were expressed at markedly higher levels than mTERT, and yet neither immortalized human fibroblasts or prevented telomere uncapping (Fig. , Table , and data not shown). These data indicate that the protein level determinants and the sequences allowing immortalization are separable. Furthermore, these data show that elevated TERT protein levels may be necessary but are not sufficient for immortalization. In addition to controlling protein level, the C-terminal domain serves a critical and species-specific function in enabling an assembled, enzymatically active telomerase complex to productively act on or to protect telomere ends.