The C-terminal extension of dyskerin is a dyskeratosis congenita mutational hotspot that modulates interaction with telomerase RNA and subcellular localization.

Dyskerin is a component of the human telomerase complex and is involved in stabilizing the human telomerase RNA (hTR). Many mutations in the DKC1 gene encoding dyskerin are found in X-linked dyskeratosis congenita (X-DC), a premature aging disorder and other related diseases. The C-terminal extension (CTE) of dyskerin contributes to its interaction with the molecular chaperone SHQ1 during the early stage of telomerase biogenesis. Disease mutations in this region were proposed to disrupt dyskerin-SHQ1 interaction and destabilize dyskerin, reducing hTR levels indirectly. However, biochemical evidence supporting this hypothesis is still lacking. In addition, the effects of many CTE disease mutations on hTR have not been examined. In this study, we tested eight dyskerin CTE variants and showed that they failed to maintain hTR levels. These mutants showed slightly reduced but not abolished interaction with SHQ1, and caused defective binding to hTR. Deletion of the CTE further reduced binding to hTR, and perturbed localization of dyskerin to the Cajal bodies and the nucleolus, and the interaction with TCAB1 as well as GAR1. Our findings suggest impaired dyskerin-hTR interaction in cells as a previously overlooked mechanism through which dyskerin CTE mutations cause X-DC and related telomere syndromes.

Orchestrating nucleic acid-protein interactions at chromosome ends: telomerase mechanisms come into focus.

Telomerase is a special reverse transcriptase ribonucleoprotein dedicated to the synthesis of telomere repeats that protect chromosome ends. Among reverse transcriptases, telomerase is unique in using a stably associated RNA with an embedded template to synthesize a specified sequence. Moreover, it is capable of iteratively copying the same template region (repeat addition processivity) through multiple rounds of RNA-DNA unpairing and reannealing, that is, the translocation reaction. Biochemical analyses of telomerase over the past 3 decades in protozoa, fungi and mammals have identified structural elements that underpin telomerase mechanisms and have led to models that account for the special attributes of telomerase. Notably, these findings and models can now be interpreted and adjudicated through recent cryo-EM structures of Tetrahymena and human telomerase holoenzyme complexes in association with substrates and regulatory proteins. Collectively, these structures reveal the intricate protein-nucleic acid interactions that potentiate telomerase's unique translocation reaction and clarify how this enzyme reconfigures the basic reverse transcriptase scaffold to craft a polymerase dedicated to the synthesis of telomere DNA. Among the many new insights is the resolution of the telomerase 'anchor site' proposed more than 3 decades ago. The structures also highlight the nearly universal conservation of a protein-protein interface between an oligonucleotide/oligosaccharide-binding (OB)-fold regulatory protein and the telomerase catalytic subunit, which enables spatial and temporal regulation of telomerase function in vivo. In this Review, we discuss key features of the structures in combination with relevant functional analyses. We also examine conserved and divergent aspects of telomerase mechanisms as gleaned from studies in different model organisms.

Complex interaction network revealed by mutation of human telomerase 'insertion in fingers' and essential N-terminal domains and the telomere protein TPP1.

In the majority of human cancer cells, cellular immortalization depends on the maintenance of telomere length by telomerase. An essential step required for telomerase function is its recruitment to telomeres, which is regulated by the interaction of the telomere protein, TPP1, with the telomerase essential N-terminal (TEN) domain of the human telomerase reverse transcriptase, hTERT. We previously reported that the hTERT 'insertion in fingers domain' (IFD) recruits telomerase to telomeres in a TPP1-dependent manner. Here, we use hTERT truncations and the IFD domain containing mutations in conserved residues or premature aging disease-associated mutations to map the interactions between the IFD and TPP1. We find that the hTERT-IFD domain can interact with TPP1. However, deletion of the IFD motif in hTERT lacking the N-terminus and the C-terminal extension does not abolish interaction with TPP1, suggesting the IFD is not essential for hTERT interaction with TPP1. Several conserved residues in the central IFD-TRAP region that we reported regulate telomerase recruitment to telomeres, and cell immortalization compromise interaction of the hTERT-IFD domain with TPP1 when mutated. Using a similar approach, we find that the IFD domain interacts with the TEN domain but is not essential for intramolecular hTERT interactions with the TEN domain. IFD-TEN interactions are not disrupted by multiple amino acid changes in the IFD or TEN, thus highlighting a complex regulation of IFD-TEN interactions as suggested by recent cryo-EM structures of human telomerase.

Long-term male-specific chronic pain via telomere- and p53­mediated spinal cord cellular senescence.

Mice with experimental nerve damage can display long­lasting neuropathic pain behavior. We show here that 4 months and later after nerve injury, male but not female mice displayed telomere length (TL) reduction and p53­mediated cellular senescence in the spinal cord, resulting in maintenance of pain and associated with decreased lifespan. Nerve injury increased the number of p53­positive spinal cord neurons, astrocytes, and microglia, but only in microglia was the increase male­specific, matching a robust sex specificity of TL reduction in this cell type, which has been previously implicated in male­specific pain processing. Pain hypersensitivity was reversed by repeated intrathecal administration of a p53­specific senolytic peptide, only in male mice and only many months after injury. Analysis of UK Biobank data revealed sex-specific relevance of this pathway in humans, featuring male­specific genetic association of the human p53 locus (TP53) with chronic pain and a male-specific effect of chronic pain on mortality. Our findings demonstrate the existence of a biological mechanism maintaining pain behavior, at least in males, occurring much later than the time span of virtually all extant preclinical studies.

Homologous recombination-mediated irreversible genome damage underlies telomere-induced senescence.

Loss of telomeric DNA leads to telomere uncapping, which triggers a persistent, p53-centric DNA damage response that sustains a stable senescence-associated proliferation arrest. Here, we show that in normal cells telomere uncapping triggers a focal telomeric DNA damage response accompanied by a transient cell cycle arrest. Subsequent cell division with dysfunctional telomeres resulted in sporadic telomeric sister chromatid fusions that gave rise to next-mitosis genome instability, including non-telomeric DNA lesions responsible for a stable, p53-mediated, senescence-associated proliferation arrest. Unexpectedly, the blocking of Rad51/RPA-mediated homologous recombination, but not non-homologous end joining (NHEJ), prevented senescence despite multiple dysfunctional telomeres. When cells approached natural replicative senescence, interphase senescent cells displayed genome instability, whereas near-senescent cells that underwent mitosis despite the presence of uncapped telomeres did not. This suggests that these near-senescent cells had not yet acquired irreversible telomeric fusions. We propose a new model for telomere-initiated senescence where tolerance of telomere uncapping eventually results in irreversible non-telomeric DNA lesions leading to stable senescence. Paradoxically, our work reveals that senescence-associated tumor suppression from telomere shortening requires irreversible genome instability at the single-cell level, which suggests that interventions to repair telomeres in the pre-senescent state could prevent senescence and genome instability.

Dyskerin: an essential pseudouridine synthase with multifaceted roles in ribosome biogenesis, splicing, and telomere maintenance.

Dyskerin and its homologs are ancient and conserved enzymes that catalyze the most common post-transcriptional modification found in cells, pseudouridylation. The resulting pseudouridines provide stability to RNA molecules and regulate ribosome biogenesis and splicing events. Dyskerin does not act independently-it is the core component of a protein heterotetramer, which associates with RNAs that contain the H/ACA motif. The variety of H/ACA RNAs that guide the function of this ribonucleoprotein (RNP) complex highlights the diversity of cellular processes in which dyskerin participates. When associated with small nucleolar (sno) RNAs, it regulates ribosomal (r) RNAs and ribosome biogenesis. By interacting with small Cajal body (sca) RNAs, it targets small nuclear (sn) RNAs to regulate pre-mRNA splicing. As a component of the telomerase holoenzyme, dyskerin binds to the telomerase RNA to modulate telomere maintenance. In a disease context, dyskerin malfunction can result in multiple detrimental phenotypes. Mutations in DKC1, the gene that encodes dyskerin, cause the premature aging syndrome X-linked dyskeratosis congenita (X-DC), a still incurable disorder that typically leads to bone marrow failure. In this review, we present the classical and most recent findings on this essential protein, discussing the evolutionary, structural, and functional aspects of dyskerin and the H/ACA RNP. The latest research underscores the role that dyskerin plays in the regulation of gene expression, translation efficiency, and telomere maintenance, along with the impacts that defective dyskerin has on aging, cell proliferation, haematopoietic potential, and cancer.

PCNA, a focus on replication stress and the alternative lengthening of telomeres pathway.

The maintenance of telomeres, which are specialized stretches of DNA found at the ends of linear chromosomes, is a crucial step for the immortalization of cancer cells. Approximately 10-15 % of cancer cells use a homologous recombination-based mechanism known as the Alternative Lengthening of Telomeres (ALT) pathway to maintain their telomeres. Telomeres in general pose a challenge to DNA replication owing to their repetitive nature and potential for forming secondary structures. Telomeres in ALT+ cells especially are subject to elevated levels of replication stress compared to telomeres that are maintained by the enzyme telomerase, in part due to the incorporation of telomeric variant repeats at ALT+ telomeres, their on average longer lengths, and their modified chromatin states. Many DNA metabolic strategies exist to counter replication stress and to protect stalled replication forks. The role of proliferating cell nuclear antigen (PCNA) as a platform for recruiting protein partners that participate in several of these DNA replication and repair pathways has been well-documented. We propose that many of these pathways may be active at ALT+ telomeres, either to facilitate DNA replication, to manage replication stress, or during telomere extension. Here, we summarize recent evidence detailing the role of PCNA in pathways including DNA secondary structure resolution, DNA damage bypass, replication fork restart, and DNA damage synthesis. We propose that an examination of PCNA and its post-translational modifications (PTMs) may offer a unique lens by which we might gain insight into the DNA metabolic landscape that is distinctively present at ALT+ telomeres.

p66ShcA potentiates the cytotoxic response of triple-negative breast cancers to PARP inhibitors.

Triple-negative breast cancers (TNBCs) lack effective targeted therapies, and cytotoxic chemotherapies remain the standard of care for this subtype. Owing to their increased genomic instability, poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are being tested against TNBCs. In particular, clinical trials are now interrogating the efficacy of PARPi combined with chemotherapies. Intriguingly, while response rates are low, cohort of patients do respond to PARPi in combination with chemotherapies. Moreover, recent studies suggest that an increase in levels of ROS may sensitize cells to PARPi. This represents a therapeutic opportunity, as several chemotherapies, including doxorubicin, function in part by producing ROS. We previously demonstrated that the p66ShcA adaptor protein is variably expressed in TNBCs. We now show that, in response to therapy-induced stress, p66ShcA stimulated ROS production, which, in turn, potentiated the synergy of PARPi in combination with doxorubicin in TNBCs. This p66ShcA-induced sensitivity relied on the accumulation of oxidative damage in TNBCs, rather than genomic instability, to potentiate cell death. These findings suggest that increasing the expression of p66ShcA protein levels in TNBCs represents a rational approach to bolster the synergy between PARPi and doxorubicin.

Regulation of human telomerase RNA biogenesis and localization.

Maintenance of telomeres is essential for genome integrity and replicative capacity in eukaryotic cells. Telomerase, the ribonucleoprotein complex that catalyses telomere synthesis is minimally composed of a reverse transcriptase and an RNA component. The sequence and structural domains of human telomerase RNA (hTR) have been extensively characterized, while the regulation of hTR transcription, maturation, and localization, is not fully understood. Here, we provide an up-to-date review of hTR, with an emphasis on current breakthroughs uncovering the mechanisms of hTR maturation and localization.

N-terminal residues of human dyskerin are required for interactions with telomerase RNA that prevent RNA degradation.

The telomerase holoenzyme responsible for maintaining telomeres in vertebrates requires many components in vivo, including dyskerin. Dyskerin binds and regulates the accumulation of the human telomerase RNA, hTR, as well as other non-coding RNAs that share the conserved H/ACA box motif. The precise mechanism by which dyskerin controls hTR levels is unknown, but is evidenced by defective hTR accumulation caused by substitutions in dyskerin, that are observed in the X-linked telomere biology disorder dyskeratosis congenita (X-DC). To understand the role of dyskerin in hTR accumulation, we analyzed X-DC substitutions K39E and K43E in the poorly characterized dyskerin N-terminus, and A353V within the canonical RNA binding domain (the PUA). These variants exhibited impaired binding to hTR and polyadenylated hTR species, while interactions with other H/ACA RNAs appear largely unperturbed by the N-terminal substitutions. hTR accumulation and telomerase activity defects of dyskerin-deficient cells were rescued by wildtype dyskerin but not the variants. hTR 3' extended or polyadenylated species did not accumulate, suggesting hTR precursor degradation occurs upstream of mature complex assembly in the absence of dyskerin binding. Our findings demonstrate that the dyskerin-hTR interaction mediated by PUA and N-terminal residues of dyskerin is crucial to prevent unchecked hTR degradation.

Zscan4 Inhibits Maintenance DNA Methylation to Facilitate Telomere Elongation in Mouse Embryonic Stem Cells.

Proper telomere length is essential for embryonic stem cell (ESC) self-renewal and pluripotency. Mouse ESCs (mESCs) sporadically convert to a transient totipotent state similar to that of two-cell (2C) embryos to recover shortened telomeres. Zscan4, which exhibits a burst of expression in 2C-like mESCs, is required for telomere extension in these cells. However, the mechanism by which Zscan4 extends telomeres remains elusive. Here, we show that Zscan4 facilitates telomere elongation by inducing global DNA demethylation through downregulation of Uhrf1 and Dnmt1, major components of the maintenance DNA methylation machinery. Mechanistically, Zscan4 recruits Uhrf1 and Dnmt1 and promotes their degradation, which depends on the E3 ubiquitin ligase activity of Uhrf1. Blocking DNA demethylation prevents telomere elongation associated with Zscan4 expression, suggesting that DNA demethylation mediates the effect of Zscan4. Our results define a molecular pathway that contributes to the maintenance of telomere length homeostasis in mESCs.

Telomerase Regulation from Beginning to the End.

The vast body of literature regarding human telomere maintenance is a true testament to the importance of understanding telomere regulation in both normal and diseased states. In this review, our goal was simple: tell the telomerase story from the biogenesis of its parts to its maturity as a complex and function at its site of action, emphasizing new developments and how they contribute to the foundational knowledge of telomerase and telomere biology.

Multiple Mechanisms Contribute to the Cell Growth Defects Imparted by Human Telomerase Insertion in Fingers Domain Mutations Associated with Premature Aging Diseases.

Normal human stem cells rely on low levels of active telomerase to sustain their high replicative requirements. Deficiency in telomere maintenance mechanisms leads to the development of premature aging diseases, such as dyskeratosis congenita and aplastic anemia. Mutations in the unique "insertion in fingers domain" (IFD) in the human telomerase reverse transcriptase catalytic subunit (hTERT) have previously been identified and shown to be associated with dyskeratosis congenita and aplastic anemia. However, little is known about the molecular mechanisms impacted by these IFD mutations. We performed comparative functional analyses of disease-associated IFD variants at the molecular and cellular levels. We report that hTERT-P721R- and hTERT-R811C-expressing cells exhibited growth defects likely due to impaired TPP1-mediated recruitment of these variant enzymes to telomeres. We showed that activity and processivity of hTERT-T726M failed to be stimulated by TPP1-POT1 overexpression and that dGTP usage by this variant was less efficient compared with the wild-type enzyme. hTERT-P785L-expressing cells did not show growth defects, and this variant likely confers cell survival through increased DNA synthesis and robust activity stimulation by TPP1-POT1. Altogether, our data suggest that multiple mechanisms contribute to cell growth defects conferred by the IFD variants.

Platinum(II) phenanthroimidazole G-quadruplex ligand induces selective telomere shortening in A549 cancer cells.

Telomere maintenance, achieved by the binding of protective shelterin capping proteins to telomeres and by either telomerase or a recombination-based alternative lengthening of telomere (ALT) mechanism, is critical for cell proliferation and survival. Extensive telomere shortening or loss of telomere integrity activates DNA damage checkpoints, leading to cell senescence or death. Although telomerase upregulation is an attractive target for anti-cancer therapy, the lag associated with telomere shortening and the potential activation of ALT pose a challenge. An alternative approach is to modify telomere interactions with binding proteins (telomere uncapping). G-quadruplex ligands stabilize structures generated from single-stranded G-rich 3'-telomere end (G-quadruplex) folding, which in principle, cannot be elongated by telomerase, thus leading to telomere shortening. Ligands can also mediate rapid anti-proliferative effects by telomere uncapping. We previously reported that the G-quadruplex ligand, phenylphenanthroimidazole ethylenediamine platinum(II) (PIP), inhibits telomerase activity in vitro[47]. In the current study, a long-term seeding assay showed that PIP significantly inhibited the seeding capacity of A549 lung cancer cells and to a lesser extent primary MRC5 fibroblast cells. Importantly, treatment with PIP caused a significant dose- and time-dependent decrease in average telomere length of A549 but not MRC5 cells. Moreover, cell cycle analysis revealed a significant increase in G1 arrest upon treatment of A549 cells, but not MRC5 cells. Both apoptosis and cellular senescence may contribute to the anti-proliferative effects of PIP. Our studies validate the development of novel and specific therapeutic ligands targeting telomeric G-quadruplex structures in cancer cells.

An intact putative mouse telomerase essential N-terminal domain is necessary for proper telomere maintenance.

BACKGROUND INFORMATION: Naturally occurring telomerase reverse transcriptase (TERT) isoforms may regulate telomerase activity, and possibly function independently of telomeres to modulate embryonic stem (ES) cell self-renewal and differentiation. RESULTS: We report the characterisation of two novel mouse TERT (mTERT) splice variants, Ins-i1[1-102] (Insi1 for short) and Del-e12[1-40] (Dele12 for short) that have not been previously described. Insi1 represents an in-frame insertion of nucleotides 1-102 from intron 1, encoding a 34 amino acid insertion at amino acid 73. Based on known functions of this region in human and Tetrahymena TERTs, the insertion interrupts the RNA interaction domain 1 implicated in low-affinity RNA binding and the telomerase essential N-terminal domain implicated in DNA substrate interactions. Dele12 contains a 40 nucleotide deletion of exon 12 which generates a premature stop codon, and possible protein lacking the C-terminus. We found Insi1 expressed in adult mouse brain and kidney and Dele12 expressed in adult mouse ovary. Dele12 was inactive in vitro and in mTERT(-/-) ES cells and Insi1 retained 26-48% of telomerase activity reconstituted by wild-type mTERT in vitro and in mTERT(-/-) ES cells. The Insi1 variant exhibited reduced DNA substrate binding in vitro and both variants exhibited a reduction in binding the telomerase RNA, mTR, when expressed in mTERT(-/-) ES cells. Stable expression of Dele12 in the mouse fibroblast CB17 cell line inhibited telomerase activity and slowed cell growth, suggesting a potential dominant-negative effect. Levels of signal-free ends, representing short telomeres, and end-to-end fusions were higher in mTERT(-/-) ES cells expressing mTERT-Insi1 and mTERT-Dele12, compared with levels observed in mTERT(-/-) ES cells expressing wild-type mTERT. In addition, in mTERT(-/-) cells expressing mTERT-Insi1, we observed chromosomes that were products of repeated breakage-bridge-fusion cycles and other telomere dysfunction-related aberrations. CONCLUSION AND SIGNIFICANCE: An intact mTERT N-terminus which contributes to mTR binding, DNA binding and telomerase activity is necessary for elongation of short telomeres and the maintenance of functional telomeres. It is reasonable to speculate that relative levels of mTERT-Insi1 may regulate telomere function in specific tissues.

The Insertion in Fingers Domain in Human Telomerase Can Mediate Enzyme Processivity and Telomerase Recruitment to Telomeres in a TPP1-Dependent Manner.

In most human cancer cells, cellular immortalization relies on the activation and recruitment of telomerase to telomeres. The telomere-binding protein TPP1 and the TEN domain of the telomerase catalytic subunit TERT regulate telomerase recruitment. TERT contains a unique domain, called the insertion in fingers domain (IFD), located within the conserved reverse transcriptase domain. We report the role of specific hTERT IFD residues in the regulation of telomerase activity and processivity, recruitment to telomeres, and cell survival. One hTERT IFD variant, hTERT-L805A, with reduced activity and processivity showed impaired telomere association, which could be partially rescued by overexpression of TPP1-POT1. Another previously reported hTERT IFD mutant enzyme with similarly low levels of activity and processivity, hTERT-V791Y, displayed defects in telomere binding and was insensitive to TPP1-POT1 overexpression. Our results provide the first evidence that the IFD can mediate enzyme processivity and telomerase recruitment to telomeres in a TPP1-dependent manner. Moreover, unlike hTERT-V791Y, hTERT-V763S, a variant with reduced activity but increased processivity, and hTERT-L805A, could both immortalize limited-life-span cells, but cells expressing these two mutant enzymes displayed growth defects, increased apoptosis, DNA damage at telomeres, and short telomeres. Our results highlight the importance of the IFD in maintaining short telomeres and in cell survival.

A novel somatic mutation in ACD induces telomere lengthening and apoptosis resistance in leukemia cells.

BACKGROUND: The identification of oncogenic driver mutations has largely relied on the assumption that genes that exhibit more mutations than expected by chance are more likely to play an active role in tumorigenesis. Major cancer sequencing initiatives have therefore focused on recurrent mutations that are more likely to be drivers. However, in specific genetic contexts, low frequency mutations may also be capable of participating in oncogenic processes. Reliable strategies for identifying these rare or even patient-specific (private) mutations are needed in order to elucidate more personalized approaches to cancer diagnosis and treatment. METHODS: Here we performed whole-exome sequencing on three cases of childhood pre-B acute lymphoblastic leukemia (cALL), representing three cytogenetically-defined subgroups (high hyperdiploid, t(12;21) translocation, and cytogenetically normal). We applied a data reduction strategy to identify both common and rare/private somatic events with high functional potential. Top-ranked candidate mutations were subsequently validated at high sequencing depth on an independent platform and in vitro expression assays were performed to evaluate the impact of identified mutations on cell growth and survival. RESULTS: We identified 6 putatively damaging non-synonymous somatic mutations among the three cALL patients. Three of these mutations were well-characterized common cALL mutations involved in constitutive activation of the mitogen-activated protein kinase pathway (FLT3 p.D835Y, NRAS p.G13D, BRAF p.G466A). The remaining three patient-specific mutations (ACD p.G223V, DOT1L p.V114F, HCFC1 p.Y103H) were novel mutations previously undescribed in public cancer databases. Cytotoxicity assays demonstrated a protective effect of the ACD p.G223V mutation against apoptosis in leukemia cells. ACD plays a key role in protecting telomeres and recruiting telomerase. Using a telomere restriction fragment assay, we also showed that this novel mutation in ACD leads to increased telomere length in leukemia cells. CONCLUSION: This study identified ACD as a novel gene involved in cALL and points to a functional role for ACD in enhancing leukemia cell survival. These results highlight the importance of rare/private somatic mutations in understanding cALL etiology, even within well-characterized molecular subgroups.

Telomere biology: Rationale for diagnostics and therapeutics in cancer.

The key step of carcinogenesis is the malignant transformation which is fundamentally a telomere biology dysfunction permitting cells to bypass the Hayflick limit and to divide indefinitely and uncontrollably. Thus all partners and structures involved in normal and abnormal telomere maintenance, protection and lengthening can be considered as potential anti-cancer therapeutic targets. In this Point of View we discuss, highlight and provide new perspectives from the current knowledge and understanding to position the different aspects of telomere biology and dysfunction as diagnostic, preventive and curative tools in the field of cancer.

Inactive C-terminal telomerase reverse transcriptase insertion splicing variants are dominant-negative inhibitors of telomerase.

Maintenance of telomere length and structure is essential for cell survival. Telomere synthesis is mediated by the ribonucleoprotein telomerase in 90% of cancer cells, and is regulated mainly by transcription of the human telomerase reverse transcriptase subunit, hTERT. However, transcriptome analysis reveals complex splicing patterns and to date, twenty-two alternatively-spliced hTERT mRNAs have been reported, yet their functions have not been fully elucidated. The best characterized hTERT spliced variants encode for inactive proteins that possess specific deletions within the hTERT catalytic domains. We studied two less well characterized hTERT splice variants (termed INS3 and 4) that encode proteins with intact reverse transcriptase motifs, but alternative C-domains due to insertion of intronic sequences. We determined the prevalence of these mRNA variants in primary cells, telomerase-positive cells and in alternative lengthening of telomere (ALT) cells and found the transcripts to be expressed mainly in telomerase-positive cell lines and to be translated into proteins as illustrated by their association with polysomes. These variants were inactive when expressed in vitro or in cells, retained DNA substrate binding in vitro but were impaired in binding the telomerase RNA component when expressed in, and immunoprecipitated from either telomerase-positive or telomerase-negative ALT cells coexpressing the telomerase RNA component. Stable expression of INS3 and INS4 variants in a hepatocarcinoma cell line inhibited telomerase activity, shortened telomeres and slowed cell growth suggesting a potential dominant-negative function.