Structure of Tetrahymena telomerase-bound CST with polymerase α-primase.

Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single-strand 3' overhang of the G-strand and, in humans, the six shelterin proteins: TPP1, POT1, TRF1, TRF2, RAP1 and TIN21,2. TPP1 and POT1 associate with the 3' overhang, with POT1 binding the G-strand3 and TPP1 (in complex with TIN24) recruiting telomerase via interaction with telomerase reverse transcriptase5 (TERT). The telomere DNA ends are replicated and maintained by telomerase6, for the G-strand, and subsequently DNA polymerase α-primase7,8 (PolαPrim), for the C-strand9. PolαPrim activity is stimulated by the heterotrimeric complex CTC1-STN1-TEN110-12 (CST), but the structural basis of the recruitment of PolαPrim and CST to telomere ends remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of Tetrahymena CST in the context of the telomerase holoenzyme, in both the absence and the presence of PolαPrim, and of PolαPrim alone. Tetrahymena Ctc1 binds telomerase subunit p50, a TPP1 orthologue, on a flexible Ctc1 binding motif revealed by cryo-EM and NMR spectroscopy. The PolαPrim polymerase subunit POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. We thus provide a snapshot of four key components that are required for telomeric DNA synthesis in a single active complex-telomerase-core ribonucleoprotein, p50, CST and PolαPrim-that provides insights into the recruitment of CST and PolαPrim and the handoff between G-strand and C-strand synthesis.

Structure of active human telomerase with telomere shelterin protein TPP1.

Human telomerase is a RNA-protein complex that extends the 3' end of linear chromosomes by synthesizing multiple copies of the telomeric repeat TTAGGG1. Its activity is a determinant of cancer progression, stem cell renewal and cellular aging2-5. Telomerase is recruited to telomeres and activated for telomere repeat synthesis by the telomere shelterin protein TPP16,7. Human telomerase has a bilobal structure with a catalytic core ribonuclear protein and a H and ACA box ribonuclear protein8,9. Here we report cryo-electron microscopy structures of human telomerase catalytic core of telomerase reverse transcriptase (TERT) and telomerase RNA (TER (also known as hTR)), and of telomerase with the shelterin protein TPP1. TPP1 forms a structured interface with the TERT-unique telomerase essential N-terminal domain (TEN) and the telomerase RAP motif (TRAP) that are unique to TERT, and conformational dynamics of TEN-TRAP are damped upon TPP1 binding, defining the requirements for recruitment and activation. The structures further reveal that the elements of TERT and TER that are involved in template and telomeric DNA handling-including the TEN domain and the TRAP-thumb helix channel-are largely structurally homologous to those in Tetrahymena telomerase10, and provide unique insights into the mechanism of telomerase activity. The binding site of the telomerase inhibitor BIBR153211,12 overlaps a critical interaction between the TER pseudoknot and the TERT thumb domain. Numerous mutations leading to telomeropathies13,14 are located at the TERT-TER and TEN-TRAP-TPP1 interfaces, highlighting the importance of TER-TERT and TPP1 interactions for telomerase activity, recruitment and as drug targets.

Structures of telomerase at several steps of telomere repeat synthesis.

Telomerase is unique among the reverse transcriptases in containing a noncoding RNA (known as telomerase RNA (TER)) that includes a short template that is used for the processive synthesis of G-rich telomeric DNA repeats at the 3' ends of most eukaryotic chromosomes1. Telomerase maintains genomic integrity, and its activity or dysregulation are critical determinants of human longevity, stem cell renewal and cancer progression2,3. Previous cryo-electron microscopy structures have established the general architecture, protein components and stoichiometries of Tetrahymena and human telomerase, but our understandings of the details of DNA-protein and RNA-protein interactions and of the mechanisms and recruitment involved remain limited4-6. Here we report cryo-electron microscopy structures of active Tetrahymena telomerase with telomeric DNA at different steps of nucleotide addition. Interactions between telomerase reverse transcriptase (TERT), TER and DNA reveal the structural basis of the determination of the 5' and 3' template boundaries, handling of the template-DNA duplex and separation of the product strand during nucleotide addition. The structure and binding interface between TERT and telomerase protein p50 (a homologue of human TPP17,8) define conserved interactions that are required for telomerase activation and recruitment to telomeres. Telomerase La-related protein p65 remodels several regions of TER, bridging the 5' and 3' ends and the conserved pseudoknot to facilitate assembly of the TERT-TER catalytic core.

Structure of human telomerase holoenzyme with bound telomeric DNA.

Telomerase adds telomeric repeats at chromosome ends to compensate for the telomere loss that is caused by incomplete genome end replication1. In humans, telomerase is upregulated during embryogenesis and in cancers, and mutations that compromise the function of telomerase result in disease2. A previous structure of human telomerase at a resolution of 8 Å revealed a vertebrate-specific composition and architecture3, comprising a catalytic core that is flexibly tethered to an H and ACA (hereafter, H/ACA) box ribonucleoprotein (RNP) lobe by telomerase RNA. High-resolution structural information is necessary to develop treatments that can effectively modulate telomerase activity as a therapeutic approach against cancers and disease. Here we used cryo-electron microscopy to determine the structure of human telomerase holoenzyme bound to telomeric DNA at sub-4 Å resolution, which reveals crucial DNA- and RNA-binding interfaces in the active site of telomerase as well as the locations of mutations that alter telomerase activity. We identified a histone H2A-H2B dimer within the holoenzyme that was bound to an essential telomerase RNA motif, which suggests a role for histones in the folding and function of telomerase RNA. Furthermore, this structure of a eukaryotic H/ACA RNP reveals the molecular recognition of conserved RNA and protein motifs, as well as interactions that are crucial for understanding the molecular pathology of many mutations that cause disease. Our findings provide the structural details of the assembly and active site of human telomerase, which paves the way for the development of therapeutic agents that target this enzyme.

A structurally conserved human and Tetrahymena telomerase catalytic core.

Telomerase is a ribonucleoprotein complex that counteracts the shortening of chromosome ends due to incomplete replication. Telomerase contains a catalytic core of telomerase reverse transcriptase (TERT) and telomerase RNA (TER). However, what defines TERT and separates it from other reverse transcriptases remains a subject of debate. A recent cryoelectron microscopy map of Tetrahymena telomerase revealed the structure of a previously uncharacterized TERT domain (TRAP) with unanticipated interactions with the telomerase essential N-terminal (TEN) domain and roles in telomerase activity. Both TEN and TRAP are absent in the putative Tribolium TERT that has been used as a model for telomerase for over a decade. To investigate the conservation of TRAP and TEN across species, we performed multiple sequence alignments and statistical coupling analysis on all identified TERTs and find that TEN and TRAP have coevolved as telomerase-specific domains. Integrating the data from bioinformatic analysis and the structure of Tetrahymena telomerase, we built a pseudoatomic model of human telomerase catalytic core that accounts for almost all of the cryoelectron microscopy density in a published map, including TRAP in previously unassigned density as well as telomerase RNA domains essential for activity. This more complete model of the human telomerase catalytic core illustrates how domains of TER and TERT, including the TEN-TRAP complex, can interact in a conserved manner to regulate telomere synthesis.

In situ imaging of intracellular human telomerase RNA with molecular beacon-functionalized gold nanoparticles.

Since the expression level of human telomerase RNA (hTR) in tumor cells is much higher than that in normal cells, the determination of hTR is of prime importance in biological research of tumors. In this work, we report molecular beacon-functionalized gold nanoparticles for hTR imaging in live cells. The molecular beacon has a loop-and-stem structure with hTR recognition sequences and a red fluorophore Cy5. In the presence of hTR, the hTR sequence could be hybridized with the loop of molecular beacon to form a duplex DNA chain and thus the fluorescence state switched from "off" to "on". After co-incubation with cells, the probe could readily permeate into cells, leading to the in situ imaging of intracellular hTR. The proposed approach could be used to differentiate tumor cells from normal ones and assess hTR expression levels in different tumor cells. Furthermore, the proposed approach allowed us to dynamically monitor the expression level of hTR in live cells and holds great potential for application in tumor diagnosis and hTR-related drug delivery.

Ultrastructural immunolocalization of telomerase and hyaluronate in migrating keratinocytes in a case of oro-pharyngeal squamous cancer.

The ultrastructural immunolocalization of telomerase and hyaluronate has been studied in a case of oropharyngeal squamous carcinoma. Immunofluorescence shows that telomerase immunolabeling is present in the cytoplasm and in nuclei of some keratinocytes during their migration into the underlying connective tissue. The electron microscope shows that the nuclear localization of telomerase mainly occurs in the large nucleoli and in likely Cajal bodies, the sites of assembling and maturation of proteins forming the telomerase complex. Aside ribosomes, the nucleolus has a role in the biosynthesis of this reverse transcriptase during cell proliferation in normal tissues and in tumors. The cytoplasmic labeling for telomerase is frequently associated with an irregular network of keratin bundles but the significance of this observation is unclear. Hyaluronate, detected through ultrastructural immunolocalization of a hyaluronate binding protein, is abundant mostly along the cell membrane of the detaching basal keratinocytes during epithelial mesenchymal transition. A coat of hyaluronate surrounds the free keratinocytes of the squamous epithelium and is present around the connective cells present underneath. The study supports the hypothesis that hyaluronate forms a pathway along which epithelial cells can migrate during epidermal mesenchymal transition and may also shield cancer cells from immune cells.

Cryo-EM structure of substrate-bound human telomerase holoenzyme.

The enzyme telomerase adds telomeric repeats to chromosome ends to balance the loss of telomeres during genome replication. Telomerase regulation has been implicated in cancer, other human diseases, and ageing, but progress towards clinical manipulation of telomerase has been hampered by the lack of structural data. Here we present the cryo-electron microscopy structure of the substrate-bound human telomerase holoenzyme at subnanometre resolution, showing two flexibly RNA-tethered lobes: the catalytic core with telomerase reverse transcriptase (TERT) and conserved motifs of telomerase RNA (hTR), and an H/ACA ribonucleoprotein (RNP). In the catalytic core, RNA encircles TERT, adopting a well-ordered tertiary structure with surprisingly limited protein-RNA interactions. The H/ACA RNP lobe comprises two sets of heterotetrameric H/ACA proteins and one Cajal body protein, TCAB1, representing a pioneering structure of a large eukaryotic family of ribosome and spliceosome biogenesis factors. Our findings provide a structural framework for understanding human telomerase disease mutations and represent an important step towards telomerase-related clinical therapeutics.

Ultrasensitive telomerase activity detection by telomeric elongation controlled surface enhanced Raman scattering.

Telomerase is now considered to be a valuable biomarker and therapeutic target in the diagnosis and treatment of cancerous diseases, which brings an urgent need in the development of fast and efficient telomerase detection strategies. Here, a new surface enhanced Raman scattering (SERS) based protocol using telomeric elongation controlled SERS (TEC-SERS) effect for the ultrasensitive telomerase detection is presented. The TEC-SERS protocol not only provides an unprecedented high sensitivity but also avoids laborious PCR procedures. The detection limit is ≈2-3 orders of magnitude lower than those of previously reported methods. This highly sensitive and straightforward TEC-SERS protocol can be developed as a routine telomerase detection method, which would greatly facilitate the telomerase based ultra-early diagnosis of malignant tumors and the fast screening of anti-cancer drugs.

The architecture of Tetrahymena telomerase holoenzyme.

Telomerase adds telomeric repeats to chromosome ends using an internal RNA template and a specialized telomerase reverse transcriptase (TERT), thereby maintaining genome integrity. Little is known about the physical relationships among protein and RNA subunits within a biologically functional holoenzyme. Here we describe the architecture of Tetrahymena thermophila telomerase holoenzyme determined by electron microscopy. Six of the seven proteins and the TERT-binding regions of telomerase RNA (TER) have been localized by affinity labelling. Fitting with high-resolution structures reveals the organization of TERT, TER and p65 in the ribonucleoprotein (RNP) catalytic core. p50 has an unanticipated role as a hub between the RNP catalytic core, p75-p19-p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution assigns function to these interactions in processive telomeric repeat synthesis. These studies provide the first view of the extensive network of subunit associations necessary for telomerase holoenzyme assembly and physiological function.

Sgs1 RecQ helicase inhibits survival of Saccharomyces cerevisiae cells lacking telomerase and homologous recombination.

In yeast telomerase mutants, the Sgs1 RecQ helicase slows the rate of senescence and also facilitates the appearance of certain types of survivors of critical telomere shortening via mechanisms dependent on Rad52-dependent homologous recombination (HR). Here we describe a third function for Sgs1 in telomerase-deficient cells, inhibition of survivors that grow independent of Rad52. Unlike tlc1 rad52 double mutants, which do not form survivors of telomere dysfunction, tlc1 rad52 sgs1 triple mutants readily generated survivors. After emerging from growth crisis, the triple mutants progressively lost telomeric and subtelomeric sequences, yet grew for more than 1 year. Analysis of cloned chromosome termini and of copy number changes of loci genome-wide using tiling arrays revealed terminal deletions extending up to 57 kb, as well as changes in Ty retrotransposon copy numbers. Amplification of the remaining terminal sequences generated large palindromes at some chromosome termini. Sgs1 helicase activity but not checkpoint function was essential for inhibiting the appearance of the survivors, and the continued absence of Sgs1 was required for the growth of the established survivors. Thus, in addition to facilitating the maintenance of telomere repeat sequences via HR-dependent mechanisms, a RecQ helicase can prevent the adoption of HR-independent mechanisms that stabilize chromosome termini without the use of natural telomere sequences. This provides a novel mechanism by which RecQ helicases may help maintain genome integrity and thus prevent age-related diseases and cancer.

Differential hTERT mRNA processing between young and older glioma patients.

The amplification of hTERT was detected in glioma tissues, although telomerase activity was not always found within these specimens. The aim of this study was to correlate the level of hTERT transcription with telomerase activity in two glioma age groups. hTERT was significantly transcribed at similar copy numbers in both age groups. However, these mRNAs translated to telomerase in 100% of the young compared to only 25% of the older patients. While hTERT transcription correlated directly to telomerase protein level and activity, as well as longer telomeres in the young group, such correlations were missing in the older group.

Telomerase as a clinical target: current strategies and potential applications.

Chromosome ends are capped by telomeres, protective DNA-protein complexes that distinguish natural ends from random DNA breaks. Telomeres erode with each successive cell division, and such divisions cease once telomeres become critically short. This proliferation limit is important as a tumor suppressive mechanism, but also contributes to the degenerative conditions associated with cellular aging. In cell types that require continuous renewal, transient expression of telomerase delays proliferation arrest by the de novo synthesis of telomere repeats. Data from our work and others' has shown that deficient telomerase activity has a negative impact on normal human physiology. In the bone marrow failure syndrome dyskeratosis congenita, telomerase enzyme deficiency leads to the premature shortening of telomeres. Premature telomere shortening most grievously affects tissues that have a rapid turnover, such as the hematopoietic and epithelial compartments. In the most severe cases, compromised renewal of hematopoietic stem cells leads to bone marrow failure and premature death. Telomerase activation/replacement shows potential as a therapy for telomere maintenance deficiency syndromes, and in tissue engineering for the degenerative conditions that are associated with normal aging. Conversely, clinical researchers are developing telomerase inhibition therapies to treat tumors, which overcome the short-telomere barrier to unrestricted proliferation by over-expressing telomerase.

Electron microscopic visualization of telomerase from Euplotes aediculatus bound to a model telomere DNA.

Binding of the telomerase ribonucleoprotein from the ciliate Euplotes aediculatus to telomeric DNA in vitro has been examined by electron microscopy (EM). Visualization of the structures that formed revealed a globular protein complex that localized to the DNA end containing the E. aediculatus telomere consensus 3'-single-strand T(4)G(4)T(4)G(4)T(4)G(2) overhang. Gel filtration confirmed that purified E. aediculatus telomerase is an active dimer in solution, and comparison of the size of the DNA-associated complex with apoferritin suggests that E. aediculatus telomerase binds to a single telomeric 3'-end as a dimer. Up to 43% of the telomerase-DNA complexes appeared by EM to involve tetramers or larger multimers of telomerase in association with two or more DNA ends. These data provide the first direct evidence that telomerase is a functional dimer and suggest that two telomerase ribonucleoprotein particles cooperate to elongate each Euplotes telomere in vivo.

Mutations in TERT, the gene for telomerase reverse transcriptase, in aplastic anemia.

BACKGROUND: Mutations in TERC, the gene for the RNA component of telomerase, cause short telomeres in congenital aplastic anemia and in some cases of apparently acquired hematopoietic failure. We investigated whether mutations in genes for other components of telomerase also occur in aplastic anemia. METHODS: We screened blood or marrow cells from 124 patients with apparently acquired aplastic anemia and 282 control subjects for sequence variations in the TERT, DKC1, NHP2, and NOP10 genes; an additional 81 patients and 246 controls were examined for genetic variations in TERT. Telomere lengths and the telomerase activity of peripheral-blood leukocytes were evaluated in patients carrying genetic variants. Identified mutations were transfected into telomerase-deficient cell lines to examine their effects and their mechanism of action on telomerase function. RESULTS: Five heterozygous, nonsynonymous mutations (which cause an amino acid change in the corresponding protein) were identified in TERT, the gene for the telomerase reverse transcriptase catalytic enzyme, among seven unrelated patients. Leukocytes from these patients had short telomeres and low telomerase enzymatic activity. In three of these patients, the mutation was also detected in buccal mucosa cells. Family members carrying the mutations also had short telomeres and reduced telomerase activity but no evident hematologic abnormality. The results of coexpression of wild-type TERT and TERT with aplastic anemia-associated mutations in a telomerase-deficient cell line suggested that haploinsufficiency was the mechanism of telomere shortening due to TERT mutations. CONCLUSIONS: Heterozygous mutations in the TERT gene impair telomerase activity by haploinsufficiency and may be risk factors for marrow failure.

Telomere higher-order structure and genomic instability.

Telomeres, nucleoprotein complexes at the end of eukaryotic chromosomes, have vital roles in chromosome integrity. Telomere chromatin structure is both intricate and dynamic allowing for a variety of responses to several stimuli. A critical determinant in telomere structure is the G-strand overhang. Facilitated by telomeric proteins, the G-strand overhang stabilizes telomere higher-order assemblies most likely by adopting unusual DNA structures. These structures influence activities that occur at the chromosome end. Dysfunctional telomeres induce signals resulting in cell growth arrest or death. To overcome telomere dysfunction, cancer cells activate the DNA polymerase, telomerase. The presence of telomerase at the telomere may establish a particular telomeric state. If the chromosome ends of cancer and normal cells exist in different states, cancer-specific telomere structures would offer a unique chemotherapeutic target.