The acquisition and maintenance of cell division potential are important characteristics of tumorigenesis. Human telomerase reverse transcriptase (TERT) and telomerase RNA (TR) can immortalize cells through telomere maintenance, and telomerase activity is one factor that contributes to the in vitro transformation of normal cells. In vitro and in vivo evidence suggest that telomerase maintains telomeres as a functional multimer. In addition, hTERT may possess telomere maintenance-independent functions. To examine the effects of hTERT loss upon an in vitro generated tumorigenic cell line we created a tumorigenic cell line from human embryonic kidney cells through expression of the SV40 early region, H-RasG12V and a Cre-mediated excisable hTERT. These immortalized cells exhibited robust anchorage-independent colony growth and tumor formation in immuno-deficient mice. Cre recombinase expression resulted in the excision of hTERT from the tumorigenic cell lines, restoring cell mortality. A return to immortality was conferred by the re-introduction of wild-type hTERT, but not with hTERT point mutations in the N-terminus (hTEN) and reverse transcriptase (RT) domains that impair in vitro telomere elongation activity. The onset of cell mortality was not immediate, and the hTERT-excised tumorigenic cells exhibited clonal variation in the anchorage-independent colony growth assay and upon tumor formation in immuno-deficient mice. We hypothesized that tumorigenic potential was not related strictly to hTERT presence, but rather telomere length and/or integrity. To investigate this possibility we maintained the tumorigenic cell lines in the continuous presence of hTERT to permit telomere elongation prior to hTERT excision; subsequently, after hTERT excision tumor formation persisted, thus demonstrating a dependence on telomere length and not hTERT presence per se. To investigate the functional multimerization of hTERT in vivo we tested if two defective hTERT polypeptides with mutations in the hTEN and RT domains could restore telomere elongation activity in vivo. Unfortunately, during the course of these experiments an unanticipated recombination event occurred that restored a catalytically active hTERT transgene. Further in vivo analysis of hTERT multimerization should be carefully designed, accounting for the selective survival advantage bestowed by wild-type hTERT. This study provides compelling evidence that hTERT does not possess telomere maintenance-independent functions.

Introduction: The effect of electrical stimulation on muscle degeneration and regeneration is largely unknown and it has not been studied in conjunction with telomeres. The consumption of non-steroidal anti-inflammatory drugs (NSAIDs) is widespread in athletes and the general population when faced with muscle soreness or injury. Furthermore, the effect of NSAIDs on muscle regeneration is controversial and its effect on telomere lengths is also unknown. Methods: Young adult males performed 200 electrically stimulated maximal isokinetic contractions with one leg (ES) and the other worked as a control (CON). They received either 1200mg ibuprofen (IBU) per day or placebo (PLA) from 21 days pre- to 30 days post-exercise. Muscle biopsies were obtained from the vastus lateralis of the CON leg at baseline (H0) and ES leg at 2.5h (H2.5) and both legs at 2, 7 and 30 days post-exercise. Blood samples were obtained at the same time points and at day 4 post-exercise. Afterwards the muscle and blood specimen were analysed for skeletal muscle and peripheral blood telomere lengths by Southern blot and signs of muscle degeneration and regeneration were quantified histologically. Results: Histological changes occurred in the ES leg, including; increased proportion of damaged myofibres (2.1±2.8%) and infiltrated myofibres (5.0±6.0%) at day 7, small myofibres (3.0±4.4%) and internally located myonuclei (2.9±3.1%) at day 30. The IBU group had significantly less internally located myonuclei at day 30 compared to PLA (1.7±2.4% vs. 4.1±3.8%). No significant differences were observed in skeletal muscle mean and minimum telomere lengths between ES and CON leg, between IBU and PLA group or between time points. Peripheral blood mean telomere lengths were not significantly different between IBU and PLA group, but between time points; H0 (9.6±1.2kb) and H2.5 (9.1±1.1kb) were significantly shorter than day 4 (10.3±1.6kb) and day 7 (10.1±1.5kb) (P<0.05). Conclusion: Electrically stimulated contractions caused significant muscle degeneration and regeneration in the 30 days post-exercise. Electrical stimulation also appeared to cause fluctuations in peripheral blood telomere lengths, but not skeletal muscle telomeres. The intake of ibuprofen appeared to interfere with muscle regeneration, but did not seem to affect the peripheral blood or skeletal muscle telomeres. However, due to marked individual variations and the small participant group it is difficult to conclude on the effects of electrical stimulation and ibuprofen on proliferative potential. Further studies are warranted to elucidate the effects of electrical stimulation and ibuprofen on blood and skeletal muscle telomeres.

Orientador: Maria Isabel Nogueira Cano / Resumo: Os parasitos do gênero Leishmania são protozoários primitivos entre os quais estão os causadores de um espectro de doenças conhecidas como leishmaniose, as quais afetam milhões de pessoas no mundo inteiro, sendo o Brasil um dos países que apresenta maior número de casos por ano. Os tratamentos e vacinas disponíveis para a leishmaniose apresentam problemas como toxicidade, alto custo e baixa eficiência, tornando importante a busca por novos alvos terapêuticos. Dada sua importância para a estabilidade genômica e proliferação celular, os telômeros têm sido considerados como alvos terapêuticos potenciais no tratamento da leishmaniose. Os telômeros são as extremidades físicas dos cromossomos lineares compostos por complexos ribonucleoproteicos que envolvem a interação entre proteínas, DNA e RNA. A maioria dos eucariotos mantêm o tamanho dos telômeros pela ação do complexo telomerase, que é minimamente composto por uma proteína (TERT, Telomerase Reverse Transcriptase) e um RNA longo não codificador (TER, Telomerase RNA). Os dois componentes do complexo telomerase em Leishmania sp. foram identificados, porém não há informação disponível sobre suas estruturas. O objetivo principal deste estudo foi a caracterização estrutural do componente TERT de Leishmania major. Utilizando alinhamentos múltiplos de sequências, foi possível verificar que os quatro domínios estruturais das telomerases canônicas estão presentes no componente TERT em Leishmania sp. Três destes domínios foram estudados ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Parasites of the Leishmania genus are primitive protozoa and among them are the causative agents of a spectrum of diseases called leishmaniasis, which affect millions of people in tropical countries worldwide, being Brazil one of the countries with higher number of cases each year. The drugs or vaccines available to treat leishmaniasis present problems such as toxicity, excessive cost a low efficiency, so it is important to search for new potential therapeutic targets. Due to their importance in genome stability and cell proliferation, telomeres have been considered a potential therapeutic target against leishmaniasis. Telomeres are the ends of linear chromosomes composed by ribonucleoproteic complexes that involve the interaction between proteins, DNA and RNA. Most eukaryotes maintain their telomere length by the action of the telomerase complex, minimally composed by a protein (TERT, Telomerase Reverse Transcriptase) and a long non-coding RNA (TER, Telomerase RNA). Both components of the Leishmania sp. telomerase complex have already been identified, however, little is known about their structure. This study had the aim to characterize the structure of the Leishmania major TERT component. Using multiple sequence alignments, we were able to verify that the four structural domains of canonical telomerases are present in Leishmania sp. Using different bioinformatic approaches three of these domains were independently studied. The TEN (Telomerase essential N-terminal) domain is... (Complete abstract click electronic access below) / Doutor

Oxidative stress is caused by an imbalance between high production of reactive oxygen species and ability of organism to detoxify them or counteract their effects. The chromosomal ends telomeres - are specialized nucleoprotein structures protecting chromosome ends from DNA damage. Telomeres of Drosophila melanogaster are arrays of specific non-long terminal repeated (LTR) retrotransposons HeT-A, TART and TAHRE. This thesis evaluates effects of exposure of paraquat, which is a strong oxidative stress inducer, on telomere length and transcription activity in Drosophila.

Orientador: Maria Isabel Nogueira Cano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T10:53:20Z (GMT). No. of bitstreams: 1 SiqueiraNeto_JairLagede_D.pdf: 2960762 bytes, checksum: 91f4b8aed472a4d4c93cf040e8c534e7 (MD5) Previous issue date: 2007 / Resumo: A leishmaniose é uma parasitose humana emergente e ainda não controlada, causada por protozoários pertencentes ao gênero Leishmania. Atualmente, a doença atinge mais de 12 milhões de pessoas, não existindo ainda métodos eficientes para seu controle e erradicação. Por estas razões a Organização Mundial da Saúde classifica a leishmaniose como doença de categoria I e incentiva o desenvolvimento de novos métodos para controlar a doença para buscar novos alvos para drogas contra parasita. No presente trabalho, estudou-se as proteínas LaRBP38 e LaRPA-1 previamente identificadas por se associarem in vitro com a simples-fita telomérica rica em ¿G¿ de L. amazonensis. Os telomeros são extremidades físicas dos cromossomos de eucariotos, formados por complexos nucleoproteicos. São responsáveis por conferir estabilidade aos cromossomos, envitando a degradação pela maquina de reparo celular e a fusão entre extremidades cromossomais. Instabilidades no telômero causam normalmente danos irreparáveis à célula podendo levar à senescência e morte celular. As proteínas que se mantém complexadas ao telômero são responsáveis por mantê-lo funcioal. Cada proteína desempenha um papel importante, seja na proteção, processo replicativo ou manutenção da estabilidade estrutural do telômero, sendo portanto, alvos potenciais para o desenvolvimento de terapias antiparasitárias. A proteína RPA é conservada em toda escala evolutiva e cumpre importantes papéis nas maquinarias de replicação, recombinação e reparo do DNA genômico ...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Leishmaniasis is an emerging and non-controlled human disease, caused by protozoan belonging to the Leishmania genera. More than 12 million people are infected and there are no efficient methods for the controlling or eradication of the disease. For those reasons, the World Health Organization classifies leishmaniasis as category I disease and encourages the development of new methods to control the disease and to find new targets for drugs against the parasite. In the present work, we studied the proteins LaRBP38 and LaRPA-1, prior identifield by in vitro assays as proteins that associates with the Leishmania amazonensis G-rich single-stranded telomeric DNA. Telomeres are the physical ends of eukaryote chromosomes formed by proteins and DNA complexes. They are responsible for the chromosome stability, avoiding degradation by the rapair machinery end-to-end fusion. Telomere instability may cause irreversible damage in the cell, leading to senescence and cell death. The proteins that interact with the telomeres are responsible for the functional dynamics of theses structures. Each protein has an important role in the protection, replication process or in the stability maintenance. Therefore, telomeric protein could be considered good targets for the development of new therapies. RPA is an evolutionary conserved protein and plays important roles in replication, recombination and repair machineries. At the telomeres, RPA recruits telomerase, the protein responsible for telomeric elengation ...Note: The complete abstract is available with the full electronic digital thesis or dissertations / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

La télomérase est essentielle pour le maintien des télomères. Elle compense le problème de réplication de l’ADN télomérique par l’ajout de séquences d’ADN aux extrémités des chromosomes. Chez l’humain, elle est très active dans les premiers stades du développement (embryon, fœtus). Puis, son activité est réprimée pour devenir indétectable dans la plupart des cellules. Ceci conduit au raccourcissement de l’ADN télomérique, à la déprotection de l’ADN et à un arrêt de division cellulaire, appelé senescence. Par contre, dans 90% des cellules de type cancéreux, elle est suractivée. Elle contribue donc à une capacité continue de la prolifération de ces cellules et à leur immortalisation. Notre organisme d’étude est la levure bourgeonnante, Saccharomyces cerevisiae. En plus de ses nombreux avantages d’utilisation, chez cette levure, la télomérase est exprimée constitutivement, ce qui signifie qu’elle nous rapproche le plus du contexte de cellule cancéreuse. Mon projet de maîtrise vise à étudier un des composants de la télomérase chez la levure S. cerevisiae, c’est-à-dire la sous-unité ARN, appelée Tlc1, et plus particulièrement une sous-partie de cet ARN, formant une jonction à trois branches (« Three Way Junction »). Jusqu’à présent, cette structure a été considérée comme étant non essentielle. Pourtant, cette structure très conservée a été démontrée comme étant essentielle à l’assemblage de la télomérase et à son activité, chez une grande variété d’espèces. Avec ce projet, j’ai tenté de déterminer si cette structure à trois branches a un quelconque rôle à jouer que ce soit dans l’assemblage ou dans l’activité de la télomérase. J’ai exploré cette structure en y réalisant des mutations et en analysant leurs effets sur la croissance cellulaire et sur la longueur des télomères. Parmi tous les mutants, la simple substitution d’un nucléotide spécifique, l’adénine 119, conduit à des télomères plus courts qui demeurent stables au fil des générations et les levures sont viables. De plus, ce raccourcissement est de l’ordre de la centaine de paires de bases lorsque la délétion d’une partie ou de la structure au complet est réalisée. C’est donc un raccourcissement significatif, représentant près d’un tiers de la longueur normale des télomères. Par ailleurs, sur des cellules présentant des télomères anormalement courts, l’ajout de ces mutations de la TWJ de TLC1 crée un phénotype létal. / Abstract : Telomerase is essential for telomere maintenance. It compensates for the End-replication problem by adding DNA sequences to the ends of chromosomes. In humans, telomerase is very active in the early stages of development (embryos, foetus). Later, its activity is repressed, and in most cells its activity becomes undetectable. This leads to telomere shortening, a deprotection of chromosome ends and to an arrest of cellular divisions, a highly regulated process also called cellular senescence. However, in cancer cells of 90% of all subtypes, telomerase is up-regulated. Hence, this enzyme promotes the proliferative capacity of cancer cells and their immortalization. The budding yeast Saccharomyces cerevisiae is our organism of study. In addition to its ease of access, telomerase is constitutively expressed in this yeast, which makes it a useful and inexpensive model of cancer cells. My master’s project aims at studying one of the telomerase components in S. cerevisiae, namely the RNA subunit Tlc1, and more specifically a part of this RNA, forming a Three-Way Junction (TWJ). So far, this structure was considered as non-essential for cell viability. However, this structure is highly conserved among species, and in diverse species it was shown to be crucial for telomerase assembly and activity. My project hence consisted in trying to determine whether or not this structure plays a role in telomerase assembly or activity. The requirements on this structure were explored by creating mutations and by analyzing their effects on cell growth and telomere length. Of all the mutants, a specific nucleotide substitution, Adenine 119 in the TWJ, leads to shortened telomeres, and this shortening is stable during further outgrowth. Furthermore, a telomere shortening of up to 100 base pairs is observed when a part or the complete TWJ structure is deleted. This shortening is quite significant as it represents about one third of the normal length of telomeres. Moreover, expressing these mutants of the TWJ in cells with short telomeres creates a synthetic lethal effect.

Le lymphome de Hodgkin est caractérisé, d’un point de vue histologique, par la présence de rares cellules tumorales nommées cellules de Reed et Sternberg, au sein d’un infiltrat cellulaire polymorphe, inflammatoire et réactionnel. Cette dernière résulte de la transformation tumorale de cellules lymphocytaires B qui acquièrent des propriétés d’échappement au système immun, de prolifération, de résistance à l’apoptose et une instabilité chromosomique. Néanmoins, la rareté des cellules tumorales, impliquant des problèmes techniques mais aussi de caractérisation des évènements primaires dans l’initiation de cette instabilité chromosomique, a été bien débattue dans la littérature. Mais les mécanismes impliqués dans l’instabilité chromosomique dans le lymphome de Hodgkin demeurent obscurs.La première partie de cette thèse a été consacrée à l’étude des mécanismes impliqués dans l’instabilité génomique du lymphome de Hodgkin via l’instabilité des microsatellites et l’instabilité chromosomique en utilisant 7 lignées de lymphome de Hodgkin. Nous avons montré pour la première fois l’implication des microsatellites dans l’instabilité génomique des lymphomes de Hodgkin (MSI-H (microsatellite instability-high) dans 3/7 lignées). De plus, nous avons montré que deux mécanismes favorisent l’émergence d’une instabilité chromosomique : le premier implique une instabilité télomérique qui est présente essentiellement dans les petites cellules tumorales induisant la formation des chromosomes dicentriques, des amplifications des gènes (Jak2 comme exemple) et des arrangements chromosomiques complexes. Le deuxième mécanisme est lié essentiellement à un défaut de réparation des cassures double-brin avec l’apparition des chromosomes dicentriques sporadiques et une fréquence importante des micronoyaux avec la formation des ponts anaphasiques.La deuxième partie de cette thèse a été consacrée à l’étude des mécanismes de maintenance des télomères dans les ganglions tumoraux du lymphome de Hodgkin (50 patients) comme dans les lignées tumorales. Nous avons montré qu’il existe une cohabitation entre les deux mécanismes importants de maintenance des télomères, l’activation de la télomérase d’une part et le mécanisme ALT (alternative lengthening of telomeres) d’autre part. Nous avons identifié la présence de petites cellules dans les ganglions hodgkiniens comme dans les lignées tumorales avec une forte activité de la télomérase par contre la cellule de Reed Sternberg est caractérisée par un profil ALT avec la présence des corps PML et une très faible activité de télomérase. La fréquence des cellules télomérase ou ALT varie d’un ganglion à un autre et d’une lignée à une autre. Un drastique raccourcissement télomérique a été observé dans les cellules exprimant la télomérase. Pour les cellules ALT, une grande hétérogénéité de la taille des télomères ainsi que la présence des chromosomes dicentriques sporadiques ont été détectées. Le suivi des patients à long terme pendant 10 ans, nous a permis d’établir une corrélation entre le profil ALT et la survenue de mortalités et de morbidités. De plus, l’étude de la radiosensibilité des lignées tumorales a montré que les lignées ALT sont plus résistantes que les lignées télomérases.La troisième partie de cette thèse a été consacrée à la validation de ces deux concepts d’instabilité chromosomique via l’instabilité télomérique et à celle des mécanismes de maintenance des télomères, en utilisant un modèle de lymphome de Hodgkin établi dans le laboratoire à partir de la lignée L428.Ces données auront une retombée clinique importante non seulement dans la compréhension et le traitement des lymphomes de Hodgkin mais aussi dans d’autres pathologies malignes. / The study of Hodgkin lymphoma (HL), with its unique microenvironment and long clinical outcomes, has provided exceptional insights into several areas of tumour biology. Findings in HL have not only improved our understanding of human carcinogenesis, but have also pioneered its translation into the clinic.Tumoral cells in HL, called Hodgkin and reed Sternberg cells (HRS), are characterized by a highly altered genomic landscape with a wide spectrum of genomic alterations, including somatic mutations, copy number alterations, complex chromosomal rearrangements, and aneuploidy. Moreover, the scarcity of HRS cells and the resulting technical problems of their in situ characterization, the primary cytogenetic events and the clonality of these possible aberrations has been a matter of debate in the past. As a consequence, a few accepted and established HL cell lines are widely used in the majority of research projects conducted worldwide.In this thesis, first we have first investigated the possible mechanisms underlying genomic instability including microsatellite and chromosomal instability in HL cell lines. We provide the first evidence that the genomic instability observed in HL is related to microsatellite instability and chromosomal instability related to two major mechanisms: first, telomere fusion leading to dicentric chromosomes formation and breakage/fusion/bridge (B/F/B) cycles involving the repeated fusion and breakage of chromosomes following the loss of telomeres in small cells associated with the lower expression of TRF2, as well as an elevated copy number of the Jak2 gene and the presence of nucleoplasmic bridges containing telomere and centromere sequences. The second mechanism is related to defective DNA repair via non homologous end-joining (NHEJ) repair with the presence of nucleoplasmic bridges without telomere or centromere sequences, accompanied by the micronucleus without centromere sequences and a higher frequency of sporadic dicentric chromosomes.The second part of this thesis has focused on investigating telomere maintenance mechanisms (TMMs) not only in HL cell lines but also in lymph nodes of HL patients. A telomerase-independent mechanism for TMM in HL has been proposed in the absence of detectable telomerase activity (TA) in some cases. The major finding of this work has been the demonstration of the presence of both telomerase and ALT mechanism in lymph nodes of HL patients as well as in HL cell lines. We have identified a subset of tumors with some small cells expressing telomerase and Reed Sternberg cells containing ALT-associated PML bodies. A significant correlation was observed between telomere length and TMMs. Drastic telomere shortening was observed in cells with telomerase expression and elevated heterogeneity of telomere length was found in ALT profile cells. Interestingly, complex chromosomal rearrangements, included sporadic dicentric formation, were observed in ALT profile cell lines. Interestingly, the relationship between TMMs and all-cause mortality and morbidity during 10 years of follow-up of HL patients using cox proportion hazard models demonstrated a poor clinical outcome for HL patients exhibiting primarily ALT mechanisms. Similarly, higher radiation sensitivity was observed for cell lines with high telomerase activity compared to cell lines with the ALT profile.

Life-history trade-offs have been well-documented within the literature through correlational and experimental studies. However, the physiological mechanisms underlying these trade-offs are less understood. Currently, there is great interest in shared mechanisms, specifically endocrine mechanisms, that might underlie the variation in life-history traits. Insulin-like growth factor-1 (IGF-1) may be one shared mechanism that is particularly important. IGF-1 is a metabolic hormone that is part of a highly conserved insulin-signaling pathway known to influence multiple life-history traits including growth and longevity across taxa, however, little is known about these trade-offs outside of laboratory populations. This dissertation focuses on the role of IGF-1 as a hormonal mechanism underlying the life-history trade-off between growth and aging in wild birds. While the causes of aging are not fully understood, telomere dynamics (length and change in length) are a potentially important mechanism underlying lifespan. To investigate the role of IGF-1 as a hormonal mechanism underlying the life-history trade-off between growth and aging in Franklin’s gulls (Leucophaeus pipixcan) and house sparrows (Passer domesticus). In Franklin’s gulls, dietary restriction reduced growth rate and IGF-1 levels but did not impact telomere dynamics. However, there was a significant negative correlation between IGF-1 levels and telomere length at the end of the post-natal growth period. In house sparrows, we found that nestling growth rates varied with respect to year, but IGF-1 levels did not. Telomere dynamics were not related to growth rates or IGF-1 levels, suggesting that during post-natal growth nestlings may be able to mitigate or even delay costs to later life stages. Finally, when exogenous IGF-1 was administered to house sparrow nestlings during the post-natal growth period, nestling growth was impacted but only in some years. Exogenous IGF-1 increased growth and final mass in 2016 and final mass in 2018. There was a trend suggesting experimental birds had shorter telomeres in 2016. Similarly, in 2018, experimental birds had significantly shorter telomeres than control birds. These effects were not observed in 2017, suggesting that trade-offs between growth an aging might only be visible under certain environmental conditions, which may vary with respect to year.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24946号 / 生博第508号 / 新制||生||68(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松田 道行, 教授 松本 智裕, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DGAM

The telomeric repeat containing RNA TERRA represents a class of long non-coding RNAs transcribed from telomeres. TERRA has been shown to play key roles in telomere maintenance. During past years it has been reported that TERRA interacts with telomerase, suggesting it may act as regulator of telomerase. However, the role of TERRA in telomerase activity in human cells is still unknown and it needs to be further investigated. Herein, we investigate the role of TERRA in telomerase activity in human cancer cells by exploiting different experimental conditions. By using single molecule RNA FISH (smiFISH) combined with confocal microscopy, we demonstrated that TERRA and the telomerase RNA subunit hTR colocalize in cancer cells. We observed that these events mainly occur in the nucleoplasm, while a fraction of TERRA transcripts colocalize with hTR molecules also at telomeres. This result suggested us the possibility of a localization-dependent role of TERRA in the regulation of telomerase activity. Surprisingly, by studying TERRA and hTR interactions during telomere length homeostasis, we observed that fewer TERRA molecules localize at chromosome ends when telomeres are elongated by telomerase. Furthermore, the fraction of telomeric TERRA-hTR colocalizing foci markedly decreased during telomere elongation. We observed that telomere shortening correlated with increased TERRA levels, in line with previously reported findings. Intriguingly, by quantifying the integrated density of the telomeric foci in smiFISH experiments combined with immunofluorescence (IF), we observed that TERRA transcripts preferentially localize to telomeres showing a brighter signal intensity as compared to the average telomeric signal of the cell, suggesting a preferential recruitment of TERRA to longer chromosome ends. These results suggest that TERRA transcripts may localize to telomeres in trans and that a displacement of TERRA from chromosome ends may be required for telomere elongation by telomerase. To gain insight into the role of TERRA in the regulation of telomerase, we used antisense oligonucleotides to deplete TERRA in cells. Interestingly, TERRA depletion resulted in increased telomerase localization to telomeres. Altogether, our findings support a model in which telomeric TERRA transcripts act as negative regulators of telomerase activity at telomeres.