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Telomeres are protective structures, composed of nucleic acids and a complex protein mixture, located at the end of the chromosomes. They play an important role in preventing genomic instability and ensuring cell health. Defects in telomere integrity result in cell dysfunction and the development of diseases, including neurodegenerative disorders, cancer and premature aging syndromes, among others. Loss of telomere integrity during normal cell aging also initiates DNA damage signals that culminate in the senescence phenotype. Fluorescence microscopy has allowed researchers to study the dynamics, shape, localization, and co-distribution of telomeres with proteins of interest. The microscopy tools to investigate these structures have evolved, making it possible to understand in greater detail the molecular mechanisms affecting telomeres that contribute to cell aging and the development of age-related diseases. Using human fibroblasts as an example, we will highlight several characteristics of telomeres that can be investigated using three different microscopy systems, including wide-field microscopy, and the two super-resolution techniques called 3D Structured Illumination Microscopy (3D-SIM) and direct Stochastic Optical Reconstruction Microscopy (dSTORM). In this review, we will also discuss their limitations and highlight their importance in answering telomere-related scientific questions.

Background Short telomeres may indicate a higher risk of cancer and other chronic diseases. Some observational studies show positive associations between leukocyte telomere length (LTL) and physical activity levels. We hypothesized, therefore, that exercise may be one strategy for slowing telomere attrition. Methods We conducted an ancillary analysis of blood from a year-long, two-centred, two-armed (1:1) randomized controlled trial of aerobic exercise versus usual inactivity. The analysis included 212 physically inactive, disease-free, non-smoking, postmenopausal women (n = 99 exercisers, n = 113 controls) in Alberta, Canada (2003–2006). The exercise prescription was aerobic exercise five days/week (supervised three days/week), 45 min/session, achieving 70–80% heart rate reserve. Baseline and 12-month LTL were analyzed using quantitative real-time polymerase chain reactions (qPCR). The primary statistical analysis was intention-to-treat, comparing the ratio of mean LTLs (12-months:baseline) for exercisers versus controls from a general linear model. Secondary analyses included a per-protocol analysis (≥90% adherence) and analyses stratified by baseline LTL, age, body mass index, and fitness level, respectively. Results Participants were overweight at baseline (mean BMI = 29 kg/m2). The primary analysis showed no evidence that LTL change differed between groups (12-month mean LTL change for the exercise group: −13% (95% CI: −32%, 11%) versus controls: −8% (95%CI: −27%, 15%); treatment effect ratio (TER, Exercise/Control) = 0.95 (95% CI: 0.68, 1.32). Per-protocol results were similar (TER = 0.87, 95% CI: 0.59, 1.30). In stratified models, TERs ranged from 0.68 to 1.35 across strata and P-interaction > 0.05). Conclusion We found no evidence to suggest that one year of aerobic exercise alters telomere attrition significantly in healthy postmenopausal women.

Telomere length and dynamics are central to understanding cell aging, genomic instability and cancer. Currently, there are limited guidelines for analyzing telomeric features in 3D using different cellular models. Image processing for telomere analysis is of increasing interest in many fields, however a lack of standardization can make comparisons and reproducibility an issue. Here we provide a user's guide for quantitative immunofluorescence microscopy of telomeres in interphase cells that covers image acquisition, processing and analysis. Strategies for determining telomere size and number are identified using normal human diploid Hs68 fibroblasts. We demonstrate how to accurately determine telomere number, length, volume, and degree of clustering using quantitative immunofluorescence. Using this workflow, we make the unexpected observation that hTERT-immortalized Hs68 cells with longer telomeres have fewer resolvable telomeres in interphase. Rigorous quantification indicates that this is due to telomeric clustering, leading to systematic underestimation of telomere number and overestimation of telomere size., Nancy Adam et al present a pipeline to analyze interphase telomeres in 3D using wide-field microscopy and image analysis. Illustrating the utility of the workflow, they detect differences in telomere length and clustering in Hs68 fibroblasts depending on whether or not cells are immortalized through hTERT expression.

Purpose A growing number of cancer survivors suffer high levels of distress, depression and stress, as well as sleep disturbance, pain and fatigue. Two different mind-body interventions helpful for treating these problems are Mindfulness-Based Cancer Recovery (MBCR) and Tai Chi/Qigong (TCQ). However, while both interventions show efficacy compared to usual care, they have never been evaluated in the same study or directly compared. This study will be the first to incorporate innovative design features including patient choice while evaluating two interventions to treat distressed cancer survivors. It will also allow for secondary analyses of which program best targets specific symptoms in particular groups of survivors, based on preferences and baseline characteristics. Methods and significance The design is a preference-based multi-site randomized comparative effectiveness trial. Participants (N = 600) with a preference for either MBCR or TCQ will receive their preferred intervention; while those without a preference will be randomized into either intervention. Further, within the preference and non-preference groups, participants will be randomized into immediate intervention or wait-list control. Total mood disturbance on the Profile of mood states (POMS) post-intervention is the primary outcome. Other measures taken pre- and post-intervention and at 6-month follow-up include quality of life, psychological functioning, cancer-related symptoms and physical functioning. Exploratory analyses investigate biomarkers (cortisol, cytokines, blood pressure/Heart Rate Variability, telomere length, gene expression), which may uncover potentially important effects on key biological regulatory and antineoplastic functions. Health economic measures will determine potential savings to the health system.

BACKGROUND: Group psychosocial interventions including mindfulness-based cancer recovery (MBCR) and supportive-expressive group therapy (SET) can help breast cancer survivors decrease distress and influence cortisol levels. Although telomere length (TL) has been associated with breast cancer prognosis, the impact of these two interventions on TL has not been studied to date. METHODS: The objective of the current study was to compare the effects of MBCR and SET with a minimal intervention control condition (a 1-day stress management seminar) on TL in distressed breast cancer survivors in a randomized controlled trial. MBCR focused on training in mindfulness meditation and gentle Hatha yoga whereas SET focused on emotional expression and group support. The primary outcome measure was relative TL, the telomere/single-copy gene ratio, assessed before and after each intervention. Secondary outcomes were self-reported mood and stress symptoms. RESULTS: Eighty-eight distressed breast cancer survivors with a diagnosis of stage I to III cancer (using the American Joint Committee on Cancer (AJCC) TNM staging system) who had completed treatment at least 3 months prior participated. Using analyses of covariance on a per-protocol sample, there were no differences noted between the MBCR and SET groups with regard to the telomere/single-copy gene ratio, but a trend effect was observed between the combined intervention group and controls (F [1,84], 3.82; P 5 .054; h 2 5.043); TL in the intervention group was maintained whereas it was found to decrease for control participants. There were no associations noted between changes in TL and changes in mood or stress scores over time. CONCLUSIONS: Psychosocial interventions providing stress reduction and emotional support resulted in trends toward TL maintenance in distressed breast cancer survivors, compared with decreases in usual care. Cancer 2014;000:000000. V C 2014 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

The noncoding RNA LINP1 acts as a scaffold that links Ku and DNA-PKcs and enables efficient DNA double-strand-break repair through nonhomologous end joining (NHEJ), thereby enhancing the resistance of triple-negative breast cancer cells to radiation and chemotherapies.

Summary Telomerase is a ribonucleoprotein reverse transcriptase (RT) that synthesizes specific DNA repeats, or telomeric DNA, at the ends of chromosomes. Telomerase is minimally composed of a protein subunit, TERT, and an RNA component, TR. Aberrant telomerase activity has been associated with most human cancers and several premature aging diseases, such as idiopathic pulmonary fibrosis (IPF), a chronic, progressive, and fatal lung disease characterized by alveolar epithelial cell damage and fibrosis. Our study focuses on three hTERT mutations that were identified in a subset of patients with IPF, in which these patients also exhibited shorter telomeres compared with age-matched controls. We characterized how three IPF-associated hTERT mutations, V144M, R865C, and R865H, affected telomerase function both in vitro and in human cells. We demonstrated that the R865 residue is crucial for repeat addition processivity and thus telomere synthesis in telomerase-positive 293 cells and telomerase-negative BJ cells, consistent with its location in the hTERT nucleotide-binding motif. In contrast, while the V144M mutant did not exhibit any biochemical defects, this mutant was unable to elongate telomeres in human cells. As a result, our studies have identified hTERT V144 and R865 as two critical residues required for proper telomerase function in cells. Together, this may explain how inherited hTERT mutations can lead to shortened telomeres in patients with IPF and, thus, provide further insight into the role of naturally occurring telomerase mutations in the pathophysiology of certain age-related disease states.

Inherited bone marrow failure (IBMF) syndromes are a group of disorders associated with insufficient production of hematopoietic cells and are characterized by a predisposition for malignancies including myelodysplastic syndrome (MDS) and acute leukemia (AML). A majority of these disorders including aplastic anemia (AA) and dyskeratosis congenita (DKC) are characterized by defects in telomere maintenance and excessively short telomeres. Studies have demonstrated an association between shortened telomeres, advanced disease and increased risk of developing blood cancers. Heterozygous mutations in the gene encoding the telomerase protein component, hTERT, are seen in 5-15% of patients with IBMF, resulting in short telomeres and advanced disease. Loss of function of one autosomal copy of hTERT is sufficient to reduce telomerase levels and accelerate telomere attrition. The degree of inactivity subsequent disease phenotype among the mutations is variable. However, the impact of telomerase mutations and shortened telomeres on disease progression and response to therapeutics is not well understood. To understand the biochemical properties and cellular consequences of mutant hTERT expression we have generated expression constructs in various cellular models in order to investigate the function of 7 distinct hTERT mutants identified in patients with AA, DKC and MDS/AML . These mutations have been identified in patients with family history or clonal evolution of disease and are all located in functionally distinct regions of the protein. Using both primary BJ fibroblasts and an in vitro reconstitution system, we have determined that each of the mutants retain varying levels of telomerase activity and repeat addition processivity, with the exception of K570N, in which both biochemical properties are severely reduced. BJ cells transfected with each of the hTERT mutants alter the growth rate compared to parental BJ cells and with the exception of K570N, bypass senescence. Expression of mutant hTERT in the THP-1 leukemic cell line results in a variety of cellular phenotypes including distinct morphological and cell cycle changes. Most notably, A202T and A1062T mutant hTERT proteins results in a delay of the G1/S transition, which may have profound implications during hematopoiesis, negatively impacting the development and differentiation of mature blood cells. In addition, we have tested the response of each of our mutant hTERT THP-1 cell lines with common chemotherapeutic agents including decitabine, etoposide, cytarabine and topotecan. While some of our mutant lines exhibit partial resistance or sensitivity to a particular agent, we have found that P704S, K1050N and A1062T exhibit global resistance to all of the agents tested compared to WT-hTERT expressing cells. Interestingly, these 3 mutants exhibit similar morphological changes, losing their ability to adhere to the culture flask. We are currently investigating the ability of these mutant cells to adhere to a human stromal layer. Finally, to address the function of mutant telomerase in hematopoiesis we are currently utilizing a long term culture method using CD34+ hematopoietic stem cells transfected with either a control vector or specific hTERT variants. This model system allows us to study the differentiation capacity of each mutant as well as examine clonal evolution. To our knowledge, this is the first long term study in CD34+ stem cells to examine the impact of hTERT mutations. CD34 cells were transfected via electroporation, sorted using fluorescent activated cell sorting using a GFP positive co-transfection vector and grown on a murine stromal feeder layer. Our results indicate the expression level of TERT is significantly increased as compared to EV indicating successful over-expression of TERT. We have been able to assess the differentiation capacity using a methocult assay as the cells age as well as examine telomere length, telomerase activity, and chromosomal stability. These experiments will determine the consequence of these hTERT mutations and telomere shortening in hematopoietic cells to help understand the contribution to bone marrow failure. By defining the role of telomeres in hematological disorders, it may be possible to alter treatment strategies based on predicted outcomes from our investigations or lead to novel therapeutics for bone marrow failure syndromes. Disclosures No relevant conflicts of interest to declare.

The Nobel Prize in Physiology or Medicine was recently awarded to Elizabeth Blackburn, Carol Greider and Jack Szostak for their pioneering studies on chromosome termini (telomeres) and their discovery of telomerase, the enzyme that synthesizes telomeres. Telomerase is a unique cellular reverse transcriptase that contains an integral RNA subunit, the telomerase RNA and a catalytic protein subunit, the telomerase reverse transcriptase (TERT), as well as several species-specific accessory proteins. Telomerase is essential for genome stability and is associated with a broad spectrum of human diseases including various forms of cancer, bone marrow failure and pulmonary fibrosis. A better understanding of telomerase structure and function will shed important insights into how this enzyme contributes to human disease. To this end, a series of high-resolution structural studies have provided critical information on TERT architecture and may ultimately elucidate novel targets for therapeutic intervention. In this review, we discuss the current knowledge of TERT structure and function, revealed through the detailed analysis of TERT from model organisms. To emphasize the physiological importance of telomeres and telomerase, we also present a general discussion of the human diseases associated with telomerase dysfunction.

Telomere integrity in human cells is maintained by the dynamic interplay between telomerase, telomere associated proteins, and DNA repair proteins. These interactions are vital to suppress DNA damage responses and unfavorable changes in chromosome dynamics. The DNA-dependent protein kinase (DNA-PK) is critical for this process. Cells deficient for functional DNA-PKcs show increased rates of telomere loss, accompanied by chromosomal fusions and translocations. Treatment of cells with specific DNA-PK kinase inhibitors leads to similar phenotypes. These observations indicate that the kinase activity of DNA-PK is required for its function at telomeres possibly through phosphorylation of essential proteins needed for telomere length maintenance. Here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a direct substrate for DNA-PK in vitro. Phosphorylation of hnRNP A1 is stimulated not only by the presence of DNA but also by the telomerase RNA component, hTR. Furthermore, we show that hnRNP A1 is phosphorylated in vivo in a DNA-PK-dependent manner and that this phosphorylation is greatly reduced in cell lines which lack hTR. These data are the first to report that hTR stimulates the kinase activity of DNA-PK toward a known telomere-associated protein, and may provide further insights into the function of DNA-PK at telomeres.

In addition to normal alternative splicing events (those that take place in untreated cells), there are also those that are inducible in response to environmental stimuli. We previously reported that the alternative splicing of p53-inducible gene 3 (PIG3) exon 4 is modulated in response to UV radiation. Here, we investigate the mechanisms mediating the alternative splicing of this exon. Through the use of various minigene constructs our results reveal that numerous factors influence the normal alternative splicing of PIG3 exon 4, and that these ultimately affect its UV-inducible alternative splicing. Included among these are sequence elements located within exon 4 itself as well as adjacent sequences required for intron definition (the pyrimidine tract, 3'- and 5'-splice sites). Within exon 4, we identified a novel hnRNP A1-dependent exonic splicing silencer (ESS) whose mutation inhibited the alternative splicing of a PIG3 minigene. Although previously implicated in the UV-inducible alternative splicing of other transcripts, RNAi-mediated silencing of hnRNP A1/A2 or hSlu7 did not prevent the UV-enhancement of exon 4 skipping. Overall, our results suggest that numerous factors contribute to the normal alternative splicing of PIG3 exon 4 and that UV-inducible increases in this process require that the splicing of this exon be maintained in a sufficiently weakened state under normal conditions.

To characterize a novel splice variant of the alpha subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMRalpha), which we discovered in human neutrophils.We used reverse transcriptase polymerase chain reaction to identify, characterize, and examine the expression of a novel splice variant of the GMRalpha transcript. At the protein level, surface plasmon resonance was used to measure the affinity of a recombinant soluble form of the novel GMRalpha protein for GM-CSF ligand. The full-length novel GMRalpha protein was expressed in a recombinant cell culture system, and its expression and localization were examined using Western blotting, I(125) GM-CSF binding assays, flow cytometry, and a soluble GMRalpha enzyme-linked immunosorbent assay.The novel GMRalpha transcript identified herein contains a previously undescribed exon of the GMRalpha gene; this exon derives from an Alu DNA repeat element, and is alternatively spliced in the novel GMRalpha transcript. Inclusion of this 102 nucleotide exon results in translation of a protein product, which we have named Alu-GMRalpha. Alu-GMRalpha is identical to cell surface GMRalpha, but additionally contains a 34 amino-acid insert in the juxtamembrane region of the extracellular domain of GMRalpha. Functionally, the Alu-GMRalpha-specific epitope does not modify the ability of the protein to bind GM-CSF, but rather appears to be preferentially targeted by ectodomain proteases to mediate the release of a third soluble GM-CSF receptor into the extracellular space.This study provides the first example of a cytokine receptor system in which soluble receptors are produced by three distinct mechanisms. Our results highlight the importance of soluble GMRalpha proteins in regulation of GM-CSF signaling.

Maintenance of telomere integrity requires the dynamic interplay between telomerase, telomere-associated proteins and DNA repair proteins. These interactions are vital to suppress DNA damage responses and changes in chromosome dynamics that can result in aneuploidy or other transforming aberrations. The interaction between the DNA repair protein Ku and the RNA component of telomerase (TLC1) in Saccharomyces cerevisiae has been shown to be important for maintaining telomere length. Here, we sought to determine whether this interaction was conserved in higher eukaryotes. Although there is no sequence similarity between TLC1 and the RNA component (hTR) of human telomerase, we show that human Ku70/80 interacts with hTR both in vitro and in a cellular context. Specifically, Ku70/80 interacts with a 47 nt region of the 3' end of hTR, which resembles the stem-loop region of the yeast Ku70/80 binding domain on TLC1. Furthermore, utilizing immunoprecipitation/RT-PCR experiments, we show that Ku interacts with hTR in cell lines deficient in the human telomerase reverse transcriptase protein (hTERT), suggesting that this interaction does not require hTERT. These data suggest that Ku interacts directly with hTR, independent of hTERT, providing evidence for the conservation of the interaction between Ku and telomerase RNA among various species and provide significant insight into how Ku is involved in telomere maintenance in higher eukaryotes.

The p53-inducible gene 3 (PIG3) is a transcriptional target of the tumor suppressor protein p53 and is thought to play a role in apoptosis. In this report, we identify a novel alternatively spliced product from the PIG3 gene that we call PIG3AS (PIG3 alternative splice). PIG3AS results from alternative pre-mRNA splicing that skips exon 4 of the five exons included in the PIG3 transcript. The resulting protein product shares its first 206 amino acids with PIG3 but has a unique 42-amino acid C terminus. In unstressed cells and after most DNA damage conditions that induce transcription from the PIG3 gene, production of the PIG3 transcript dominates. However, in response to UV light, pre-mRNA splicing shifts dramatically in favor of PIG3AS. Unlike the PIG3 protein, the PIG3AS protein is rapidly degraded with a short half-life and is stabilized by proteasome inhibition. Our results illustrate the first example of an endogenous, UV-inducible, alternative splicing event and that control of the splicing machinery is involved in the cellular DNA damage response. They also suggest that rapid proteolytic degradation represents a cellular mechanism for uncoupling p53 activity from PIG3 gene activation that is independent of promoter selectivity.

The catalytic subunit of telomerase, the telomerase reverse transcriptase (TERT), possesses the hallmark amino acid motifs of a reverse transcriptase (RT) (23, 29, 40, 47). However, unlike viral RTs, telomerase is a unique eukaryotic RT that carries an intrinsic RNA template essential for the de novo addition of telomere sequences (reviewed in reference 19). Proteins associated with telomerase activity include TEP1 (22, 48), hsp90/p23 (18, 25), dyskerin (42, 43), L22 (32), and hStau (32) in mammals; the Sm proteins (54) as well as Est1p and Est3p, (26, 56) in Saccharomyces cerevisiae; and p80, p95, and p43 in ciliates (1, 11, 21, 35). TEP1 is not essential for telomerase activity in vitro or in vivo (5, 37). A subset of these associated factors are known to serve distinct roles in telomerase assembly and telomere length maintenance (15, 16, 27, 34, 41, 43, 51, 54). In S. cerevisiae, different telomerase RNAs can functionally cooperate to form an active telomerase complex in vivo. Prescott and Blackburn demonstrated the presence of at least two primer recognition-elongation sites within S. cerevisiae telomerase (50). In addition, they showed that a mutant telomerase RNA incapable of telomere elongation could nonetheless support elongation in a diploid strain containing one mutant and one wild-type telomerase RNA (50). These results provided the first evidence that telomerase could form an active multimer in vivo that might contain, at minimum, two active sites (50). A recombinant reconstitution assay for human telomerase showed that two separately inactive, nonoverlapping fragments of human telomerase RNA could reconstitute telomerase activity in vitro (59). While consistent with a model of telomerase RNA multimerization, these results are also consistent with reconstitution of a single active telomerase RNA from two inactive telomerase RNA fragments. In vitro, the minimal requirements for telomerase activity appear to comprise the telomerase RNA and human TERT (hTERT) (3, 6, 9, 60). Previously, we found that the first 300 amino acid residues (aa) of hTERT were dispensable for telomerase activity in vitro and in vivo (5) (summarized in Fig. ​Fig.1A).1A). However, the telomerase activities associated with N-terminal truncations of hTERT were severely reduced in rabbit reticulocyte lysates (RRLs) relative to the activities achieved when the same hTERT truncation proteins were introduced into telomerase-positive 293T cells (5). Furthermore, deletion of the C-terminal 204 aa of hTERT did not affect telomerase activity in 293T cells, whereas all but the C-terminal 20 aa are absolutely required for telomerase activity in RRL (4, 5) (summarized in Fig. ​Fig.1A).1A). In this study, we set out to determine whether the observed discrepancies in activity between truncated hTERT proteins in RRL and 293T cells might be explained by the multimerization of specific hTERT fragments with endogenous hTERT. FIG. 1 Physical and functional interactions of full-length and truncated hTERT proteins in vitro. (A) A schematic diagram of full-length hTERT, including a summary of the minimal fragments of hTERT that are sufficient for telomerase activity in RRL and 293T ...

The minimal, active core of human telomerase is postulated to contain two components, the telomerase RNA hTER and the telomerase reverse transcriptase hTERT. The reconstitution of human telomerase activity in vitro has facilitated the identification of sequences within the telomerase RNA and the RT motifs of hTERT that are essential for telomerase activity. However, the precise role of residues outside the RT domain of hTERT is unknown. Here we have delineated several regions within hTERT that are important for telomerase catalysis, primer use, and interaction with the telomerase RNA and the telomerase-associated protein TEP1. In particular, certain deletions of the amino and carboxy terminus of hTERT that retained an interaction with telomerase RNA and TEP1 were nonetheless completely inactive in vitro and in vivo. Furthermore, hTERT truncations lacking the amino terminus that were competent to bind the telomerase RNA were severely compromised for the ability to elongate telomeric and nontelomeric primers. These results suggest that the interaction of telomerase RNA with hTERT can be functionally uncoupled from polymerization, and that there are regions outside the RT domain of hTERT that are critical for telomerase activity and primer use. These results establish that the human telomerase RT possesses unique polymerization determinants that distinguish it from other RTs.

We have used ethylation protection experiments and modification interference using phosphorothioate nucleosides to identify phosphate groups involved in the magnesium-dependent tertiary structure and function of the VS ribozyme, a small, self-cleaving RNA. Phosphorothioate interference-rescue experiments in the presence of the thiophilic manganese ion implicate four phosphate groups in direct metal ion binding. Phosphorothioate substitution also creates a new manganese binding site that increases the cis cleavage rate of the ribozyme, possibly by disrupting an inhibitory structure. Interpreting these data in the context of a recently developed structural model shows that almost all of the important phosphate groups are located in the central core of the ribozyme. The model suggests roles for certain phosphate groups in particular steps of RNA folding and identifies a candidate region for the active site of the ribozyme.

Telomerase is a ribonucleoprotein enzyme complex that adds single-stranded telomere DNA to chromosome ends [1]. The RNA component of telomerase contains the template for telomeric DNA addition and is essential for activity [1,2]. Telomerase proteins have been identified in ciliates, yeast and mammals [3–12]. In Saccharomyces cerevisiae , the Est2 protein is homologous to the 123 kDa reverse transcriptase subunit of Euplotes telomerase, and is essential for telomerase activity [8]. In humans, telomerase activity is associated with the telomerase RNA hTR [13], the telomerase RNA-binding protein TP1/TLP1 [5,12] and the TP2 protein encoded by the human EST2 homolog [12] (also known as TRT1, hEST2 or TCS1 [9–11]). The minimal complex sufficient for activity is, however, unknown. We have reconstituted human telomerase activity in reticulocyte lysates and find that only exogenous hTR and TP2 are required for telomerase activity in vitro . Recognition of telomerase RNA by TP2 was species specific, and nucleotides 10–159 of hTR were sufficient for telomerase activity. Telomerase activity immunoprecipitated from the reticulocyte lysate contained hTR and recombinant TP2. Substitution of conserved amino acid residues in the reverse transcriptase domain of TP2 completely abolished telomerase activity. We suggest that TP2 and hTR might represent the minimal catalytic core of human telomerase.

Telomerase is a ribonucleoprotein reverse transcriptase (RT) that synthesizes specific DNA repeats, or telomeric DNA, at the ends of chromosomes. Telomerase is minimally composed of a protein subunit, TERT, and an RNA component, TR. Aberrant telomerase activity has been associated with most human cancers and several premature aging diseases, such as idiopathic pulmonary fibrosis (IPF), a chronic, progressive, and fatal lung disease characterized by alveolar epithelial cell damage and fibrosis. Our study focuses on three hTERT mutations that were identified in a subset of patients with IPF, in which these patients also exhibited shorter telomeres compared with age-matched controls. We characterized how three IPF-associated hTERT mutations, V144M, R865C, and R865H, affected telomerase function both in vitro and in human cells. We demonstrated that the R865 residue is crucial for repeat addition processivity and thus telomere synthesis in telomerase-positive 293 cells and telomerase-negative BJ cells, consistent with its location in the hTERT nucleotide-binding motif. In contrast, while the V144M mutant did not exhibit any biochemical defects, this mutant was unable to elongate telomeres in human cells. As a result, our studies have identified hTERT V144 and R865 as two critical residues required for proper telomerase function in cells. Together, this may explain how inherited hTERT mutations can lead to shortened telomeres in patients with IPF and, thus, provide further insight into the role of naturally occurring telomerase mutations in the pathophysiology of certain age-related disease states.

Telomerase is a ribonucleoprotein reverse transcriptase (RT) that processively synthesizes telomeric repeats onto the ends of linear chromosomes to maintain genomic stability. It has been proposed that the N terminus of the telomerase protein subunit, telomerase RT (TERT), contains an anchor site that forms stable interactions with DNA to prevent enzyme-DNA dissociation during translocation and to promote realignment events that accompany each round of telomere synthesis. However, it is not known whether human TERT (hTERT) can directly interact with DNA in the absence of the telomerase RNA subunit. Here we use a novel primer binding assay to establish that hTERT forms stable and specific contacts with telomeric DNA in the absence of the human telomerase RNA component (hTR). We show that hTERT-mediated primer binding can be functionally uncoupled from telomerase-mediated primer extension. Our results demonstrate that the first 350 amino acids of hTERT have a critical role in regulating the strength and specificity of protein-DNA interactions, providing additional evidence that the TERT N terminus contains an anchor site. Furthermore, we establish that the RT domain of hTERT mediates important protein-DNA interactions. Collectively, these data suggest that hTERT contains distinct anchor regions that cooperate to help regulate telomerase-mediated DNA recognition and elongation.

In eukaryotic cells, the ends of linear chromosomes are maintained by nucleoprotein complexes called telomeres, which are composed of long stretches of repetitive DNA sequences bound by specific telomere binding proteins. The repetitive DNA sequence (repeats of TTAGGG in humans) is composed of double stranded DNA and, at its extreme end, a single-stranded G-rich 3' overhang, referred to as the G-tail. The telomere folds back on itself to form a lariat-shaped loop called the T (for telomere) loop, and the G-tail invades the double stranded DNA of the T-loop to form a D (for displacement) loop. This entire structure is stabilized by interaction with proteins of the shelterin complex, namely TRF1, TRF2, TIN2, RAP1, TPP1 and POT1 [1,2]. The T-loop structure is thought to protect the ends of linear chromosomes from being recognized by the cell as DNA double strand breaks (DSBs) [3]. Paradoxically, numerous proteins known to be involved in the detection and repair of DSBs are also found at telomeres. One such protein is the DNA-dependent protein kinase catalytic subunit, DNA-PKcs, which plays a critical role in the repair of DSBs by non-homologous end-joining in mammalian cells [4,5]. Another is the Werner Syndrome protein, WRN, a multifunctional protein with 3'-5' DNA helicase and 3'-5' exonuclease activities [6]. Both DNA-PKcs and WRN have functions not only in DNA repair and maintenance of genomic stability, but also in the maintenance of telomere integrity. Loss of DNA-PKcs results in telomere uncapping and chromosomal fusions [7], whereas loss of WRN results in telomere shortening and is associated with a premature aging phenotype [6]. One of the current challenges in telomere biology is to understand how these proteins and protein-DNA complexes regulate telomere length, maintain telomere integrity, and prevent the initiation of a DNA damage response and cellular senescence. In this issue of Aging, Kusumoto-Matsuo and colleagues [8] report that DNA-PKcs stimulates the helicase activity of WRN towards model D loop structures in vitro. DNA-PKcs did not affect the exonuclease activity of WRN or the unwinding activity of the related RecQ helicase, BLM, suggesting that this effect is specific for the WRN helicase activity. Significantly, WRN and DNA-PKcs were shown to cooperate to protect the 3' single stranded ends of telomeres in vivo by preventing G-tail shortening. These findings have important implications for how DNA-PKcs and WRN cooperate to resolve these complex telomeric DNA structures. Moreover, since shortening of the G-overhang contributes to onset of cellular senescence [9], these findings suggest that the WRN/DNA-PKcs interaction might contribute to preventing cellular senescence. This study is the first to report a functional interaction between WRN and DNA-PKcs at telomeres and provides a stepping-stone to further elucidate the critical roles that these proteins play in telomere integrity and cellular senescence. It also raises some interesting questions. For example, it will be interesting to determine whether other proteins present at telomeres such as TRF2, which has recently been shown to regulate the activity of WRN [10], affects the regulation of WRN by DNA-PKcs. Equally interesting will be to determine the molecular mechanism by which DNA-PKcs stimulates the helicase activity of WRN and whether the DNA-PKcs binding partner, Ku, plays a role in this process. It will also be interesting to determine whether WRN-mediated unwinding promotes telomerase accessibility to the telomere for example during S-phase. Interestingly, the authors have also shown that DNA-PKcs stimulates the activity of WRN towards D-loops composed of non-telomeric DNA sequences, suggesting that these findings may extend to D-loop structures formed during other nuclear processes such as homologous recombination. In conclusion, these findings bring us one step closer to unraveling the complex interplay between DNA damage response proteins and telomeres and to understanding how these complex and dynamic structures contribute to the maintenance of genomic stability and the prevention of premature aging.

Varkud Satellite (VS) RNA contains a small self-cleaving RNA motif that is distinct in its sequence and secondary structure from the hammerhead, hairpin, and hepatitis delta virus ribozymes, which are found in other natural RNAs. We have used a base specific chemical damage selection (modification interference) assay to identify functionally important nucleotides and structural elements in VS RNA. Many modified bases interfered with self-cleavage and most of these clustered at helix junctions, certain internal loops, and in a long-range pseudoknot; these correspond to previously determined sites of magnesium-dependent protection from chemical modification. The clustering suggests that these bases are important not only for a large number of individual interactions, but because they form a smaller number of structural elements that are important for activity. Modification of bases in other single-stranded regions, which did not exhibit magnesium-dependent protection, generally did not interfere with activity, suggesting that some of these regions might be dispensable for function. Surprisingly, we found a separate cluster of bases whose modification significantly enhanced cleavage. These bases appear to form a structural element that naturally attenuates the self-cleavage reaction. In natural VS RNA this attenuator structure may affect the cleavage/ligation equilibrium by inhibiting circle re-opening, thereby helping to maintain the RNA in a circular form, which is the predominant form of VS RNA in vivo. Taken together, the results of the damage selection experiments localize the catalytic core of VS RNA to a small subset of the previously determined minimal contiguous sequence.

The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key player in the processes of hematopoiesis and inflammation. The functional effects of GM-CSF are mediated by binding of the cytokine to the cell surface GM-CSF receptor, which is comprised of a ligand-binding alpha subunit (GMRα) and a signal-transducing beta subunit (GMRβ). In addition, there are at least 2 soluble GMRα isoforms (sGMRα)-one generated by alternative splicing, and a second produced by proteolytic release of the GMRα extracellular domain from the cell surface. While examining expression of the spliced soluble and transmembrane (tmGMRα) isoforms of GMRα in neutrophils by RT-PCR, we detected a third, higher molecular weight, GMRα transcript. This was surprising since the tmGMRα transcript is comprised of all 13 exons of the published GMRα gene structure (Nakagawa et al., 1994). We therefore hypothesized that we had discovered a previously undescribed exon of the GMRα gene. The novel GMRα (nGMRα) transcript was subsequently cloned from human neutrophils. Sequencing of nGMRα indicated inclusion of a 102 nucleotide sequence between exons 10 and 11 of the GMRα gene. Interestingly, the novel exon and surrounding intronic sequence appear to represent an Alu-repeat element, indicating that this exon is likely primate-specific. Segments of these repetitive DNA elements become inserted into mature mRNAs by way of splicing in a process termed “exonization”. In fact, it now seems that more than 5% of the alternatively spliced exons in the human genome are Alu-derived. At the protein level, this novel transcript is predicted to contain all 400 amino acids of the tmGMRα protein as well as an additional 34 amino acids within the membrane-proximal region of the extracellular domain. We have expressed the novel GMRα clone, as well as a soluble version of novel GMRα (sol-nGMRα), in the murine factor-dependent cell line Ba/F3. The sol-nGMRα protein is secreted from these cells into conditioned media, as expected. We have purified sol-nGMRα protein and used it to determine the affinity of nGMRα for GM-CSF. By flow cytometry, we were unable to detect full-length nGMRα on the surface of Ba/F3 cells, but we were able to detect nGMRα in conditioned media from nGMRα-expressing Ba/F3 cells. In addition, while tmGMRα-expressing Ba/F3 cells are able to proliferate in response to human GM-CSF in the absence of murine growth factors, the nGMRα-expressing Ba/F3 cells do not respond to GM-CSF. Our results indicate that the full-length nGMRα protein is proteolytically cleaved from the cell surface or alternatively, that its subcellular localization is otherwise disrupted. As the nGMRα transcript was first detected in neutrophils isolated from a human donor, we sought to determine whether nGMRα would be expressed by other donors and in other hematopoietic cell types. Our results indicate that all donors tested express nGMRα, and that nGMRα is present in all hematopoietic cell types expressing the tmGMRα transcript, although tmGMRα nonetheless represents the predominant transcript. It is becoming increasingly clear that GM-CSF signaling is more complex than was previously thought. In order to understand the actions of GM-CSF in the clinical setting, it is critical that we first define all components of the GM-CSF signaling axis.