Telomerase, telomeric DNA and associated proteins together represent a complex, finely tuned and functionally conserved mechanism that ensures genome integrity by protecting and maintaining chromosome ends. Changes in its components can threaten an organism's viability. Nevertheless, molecular innovation in telomere maintenance has occurred multiple times during eukaryote evolution, giving rise to species/taxa with unusual telomeric DNA sequences, telomerase components or telomerase-independent telomere maintenance. The central component of telomere maintenance machinery is telomerase RNA (TR) as it templates telomere DNA synthesis, its mutation can change telomere DNA and disrupt its recognition by telomere proteins, thereby leading to collapse of their end-protective and telomerase recruitment functions. Using a combination of bioinformatic and experimental approaches, we examine a plausible scenario of evolutionary changes in TR underlying telomere transitions. We identified plants harbouring multiple TR paralogs whose template regions could support the synthesis of diverse telomeres. In our hypothesis, formation of unusual telomeres is associated with the occurrence of TR paralogs that can accumulate mutations, and through their functional redundancy, allow for the adaptive evolution of the other telomere components. Experimental analyses of telomeres in the examined plants demonstrate evolutionary telomere transitions corresponding to TR paralogs with diverse template regions.

• Klíčová slova
• evolution, land plants, mechanism of telomere transitions, telomerase RNA paralogs, unusual telomere DNA,
• MeSH
• RNA genetika   metabolismus   MeSH
• rostliny metabolismus   MeSH
• telomerasa * genetika   metabolismus   MeSH
• telomery genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA MeSH
• telomerasa * MeSH
• telomerase RNA MeSH Prohlížeč

Telomere maintenance is a highly coordinated process, and its misregulation is linked to cancer and telomere-shortening syndromes. Recent studies have shown that the TEL-patch--a cluster of amino acids on the surface of the shelterin component TPP1--is necessary for the recruitment of telomerase to the telomere in human cells. However, there has been only basic biochemical analysis of the role of TPP1 in the telomerase recruitment process. Here we develop an in vitro assay to quantitatively measure the contribution of the TEL-patch to telomerase recruitment--binding and extension of the first telomeric repeat. We also demonstrate that the TEL-patch contributes to the translocation step of the telomerase reaction. Finally, our quantitative observations indicate that the TEL-patch stabilizes the association between telomerase and telomeric DNA substrates, providing a molecular explanation for its contributions to telomerase recruitment and action.

• Klíčová slova
• DNA substrate-competition, TPP1 TEL-patch, processivity, telomerase recruitment, telomere,
• MeSH
• aminokyseliny metabolismus   MeSH
• aminopeptidasy genetika   metabolismus   MeSH
• dipeptidylpeptidasy a tripeptidylpeptidasy genetika   metabolismus   MeSH
• DNA metabolismus   MeSH
• HEK293 buňky MeSH
• kompetitivní vazba MeSH
• lidé MeSH
• molekulární modely MeSH
• proteiny vázající telomery chemie   metabolismus   MeSH
• replikace DNA * MeSH
• retardační test MeSH
• serinové proteasy genetika   metabolismus   MeSH
• shelterinový komplex chemie   metabolismus   MeSH
• telomerasa genetika   metabolismus   MeSH
• telomery genetika   metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• ACD protein, human MeSH Prohlížeč
• aminokyseliny MeSH
• aminopeptidasy MeSH
• dipeptidylpeptidasy a tripeptidylpeptidasy MeSH
• DNA MeSH
• proteiny vázající telomery MeSH
• serinové proteasy MeSH
• shelterinový komplex MeSH
• telomerasa MeSH
• TERT protein, human MeSH Prohlížeč

Despite the essential requirement of telomeric DNA for genome stability, the length of telomere tracts between species substantially differs, raising the question of the minimal length of telomeric DNA necessary for proper function. Here, we address this question using a hypomorphic allele of the telomerase catalytic subunit, TERT. We show that although this construct partially restored telomerase activity to a tert mutant, telomeres continued to shorten over several generations, ultimately stabilizing at a bimodal size distribution. Telomeres on two chromosome arms were maintained at a length of 1 kb, while the remaining telomeres were maintained at 400 bp. The longest telomeres identified in this background were also significantly longer in wild-type populations, suggesting cis-acting elements on these arms either promote telomerase processivity or recruitment. Genetically disrupting telomerase processivity in this background resulted in immediate lethality. Thus, telomeres of 400 bp are both necessary and sufficient for Arabidopsis viability. As this length is the estimated minimal length for t-loop formation, our data suggest that telomeres long enough to form a t-loop constitute the minimal functional length.

• Klíčová slova
• genome stability, subtelomere, t-loop, telomerase, telomeres,
• MeSH
• Arabidopsis MeSH
• homeostáza telomer * MeSH
• mutace MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• telomerasa genetika   metabolismus   MeSH
• telomery genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• proteiny huseníčku MeSH
• telomerasa MeSH
• TERT protein, Arabidopsis MeSH Prohlížeč

The repetitive telomeric DNA at chromosome ends is protected from unwanted repair by telomere-associated proteins, which form the shelterin complex in mammals. Recent works have provided new insights into the mechanisms of how human shelterin assembles and recruits telomerase to telomeres. Inhibition of telomerase activity and telomerase recruitment to chromosome ends is a promising target for anticancer therapy. Here, we summarize results of quantitative assessments and newly emerged structural information along with the status of the most promising approaches to telomerase inhibition in cancer cells. We focus on the mechanism of shelterin assembly and the mechanisms of how shelterin affects telomerase recruitment to telomeres, addressing the conceptual dilemma of how shelterin allows telomerase action and regulates other essential processes. We evaluate how the identified critical interactions of telomerase and shelterin might be elucidated in future research of new anticancer strategies.

• Klíčová slova
• anticancer, assembly, inhibitor, protein-DNA interaction, protein-protein interaction, quantitative biology, shelterin, telomerase, telomere,
• MeSH
• antitumorózní látky farmakologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• lidé MeSH
• proteiny vázající telomery chemie   metabolismus   MeSH
• shelterinový komplex MeSH
• telomerasa antagonisté a inhibitory   chemie   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• antitumorózní látky MeSH
• inhibitory enzymů MeSH
• proteiny vázající telomery MeSH
• shelterinový komplex MeSH
• telomerasa MeSH

Telomerase maturation and recruitment to telomeres is regulated by several telomerase- and telomere-associated proteins. Among a number of proteins, human Pontin and Reptin play critical roles in telomerase biogenesis. Here we characterized plant orthologues of Pontin and Reptin, RuvBL1 and RuvBL2a, respectively, and show association of Arabidopsis thaliana RuvBL1 (AtRuvBL1) with the catalytic subunit of telomerase (AtTERT) in the nucleolus in vivo. In contrast to mammals, interactions between AtTERT and AtRuvBL proteins in A. thaliana are not direct and they are rather mediated by one of the Arabidopsis thaliana Telomere Repeat Binding (AtTRB) proteins. We further show that plant orthologue of dyskerin, named AtCBF5, is indirectly associated with AtTRB proteins but not with the AtRuvBL proteins in the plant nucleus/nucleolus, and interacts with the Protection of telomere 1 (AtPOT1a) in the nucleolus or cytoplasmic foci. Our genome-wide phylogenetic analyses identify orthologues in RuvBL protein family within the plant kingdom. Dysfunction of AtRuvBL genes in heterozygous T-DNA insertion A. thaliana mutants results in reduced telomerase activity and indicate the involvement of AtRuvBL in plant telomerase biogenesis.

• Klíčová slova
• Arabidopsis, AtPOT1a, AtRuvBL, AtTERT, AtTRB, Pontin, Reptin, nucleolus, telomerase assembly,
• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• buněčné jadérko metabolismus   MeSH
• DNA-helikasy metabolismus   MeSH
• fylogeneze MeSH
• jaderné proteiny MeSH
• katalytická doména MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteiny vázající RNA metabolismus   MeSH
• proteiny vázající telomery metabolismus   MeSH
• telomerasa metabolismus   MeSH
• telomery metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-helikasy MeSH
• jaderné proteiny MeSH
• NAP57 protein, Arabidopsis MeSH Prohlížeč
• POT1A protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• proteiny vázající RNA MeSH
• proteiny vázající telomery MeSH
• telomerasa MeSH
• TERT protein, Arabidopsis MeSH Prohlížeč
• TRB3 protein, Arabidopsis MeSH Prohlížeč

The life cycle of telomerase involves dynamic and complex interactions between proteins within multiple macromolecular networks. Elucidation of these associations is a key to understanding the regulation of telomerase under diverse physiological and pathological conditions from telomerase biogenesis, through telomere recruitment and elongation, to its non-canonical activities outside of telomeres. We used tandem affinity purification coupled to mass spectrometry to build an interactome of the telomerase catalytic subunit AtTERT, using Arabidopsis thaliana suspension cultures. We then examined interactions occurring at the AtTERT N-terminus, which is thought to fold into a discrete domain connected to the rest of the molecule via a flexible linker. Bioinformatic analyses revealed that interaction partners of AtTERT have a range of molecular functions, a subset of which is specific to the network around its N-terminus. A significant number of proteins co-purifying with the N-terminal constructs have been implicated in cell cycle and developmental processes, as would be expected of bona fide regulatory interactions and we have confirmed experimentally the direct nature of selected interactions. To examine AtTERT protein-protein interactions from another perspective, we also analysed AtTERT interdomain contacts to test potential dimerization of AtTERT. In total, our results provide an insight into the composition and architecture of the plant telomerase complex and this will aid in delineating molecular mechanisms of telomerase functions.

• Klíčová slova
• AtPOT1a, PURα1, Pontin, Reptin, TAP-MS, Telomerase,
• MeSH
• Arabidopsis enzymologie   genetika   MeSH
• buněčné jádro enzymologie   MeSH
• chromatografie afinitní MeSH
• exprese genu MeSH
• interakční proteinové domény a motivy MeSH
• kultivované buňky MeSH
• mapování interakce mezi proteiny MeSH
• mapy interakcí proteinů MeSH
• multimerizace proteinu MeSH
• proteiny huseníčku genetika   izolace a purifikace   metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• telomerasa genetika   izolace a purifikace   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny huseníčku MeSH
• telomerasa MeSH

Human telomeric repeat binding factors TRF1 and TRF2 along with TIN2 form the core of the shelterin complex that protects chromosome ends against unwanted end-joining and DNA repair. We applied a single-molecule approach to assess TRF1-TIN2-TRF2 complex formation in solution at physiological conditions. Fluorescence cross-correlation spectroscopy was used to describe the complex assembly by analyzing how coincident fluctuations of differently labeled TRF1 and TRF2 correlate when they move together through the confocal volume of the microscope. We observed, at the single-molecule level, that TRF1 effectively substitutes TRF2 on TIN2. We assessed also the effect of another telomeric factor TPP1 that recruits telomerase to telomeres. We found that TPP1 upon binding to TIN2 induces changes that expand TIN2 binding capacity, such that TIN2 can accommodate both TRF1 and TRF2 simultaneously. We suggest a molecular model that explains why TPP1 is essential for the stable formation of TRF1-TIN2-TRF2 core complex.

• Klíčová slova
• TIN2, assembly, shelterin, single-molecule, telomere,
• MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• protein TRF1 genetika   metabolismus   MeSH
• protein TRF2 genetika   metabolismus   MeSH
• proteinové domény MeSH
• proteiny vázající telomery genetika   metabolismus   MeSH
• shelterinový komplex * metabolismus   MeSH
• telomerasa metabolismus   MeSH
• telomery metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ACD protein, human MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• protein TRF1 MeSH
• protein TRF2 MeSH
• proteiny vázající telomery MeSH
• shelterinový komplex * MeSH
• telomerasa MeSH
• TERF2 protein, human MeSH Prohlížeč
• TINF2 protein, human MeSH Prohlížeč

BACKGROUND: Canonical telomeres (telomerase-synthetised) are readily forming G-quadruplexes (G4) on the G-rich strand. However, there are examples of non-canonical telomeres among eukaryotes where telomeric tandem repeats are invaded by specific retrotransposons. Drosophila melanogaster represents an extreme example with telomeres composed solely by three retrotransposons-Het-A, TAHRE and TART (HTT). Even though non-canonical telomeres often show strand biased G-distribution, the evidence for the G4-forming potential is limited. RESULTS: Using circular dichroism spectroscopy and UV absorption melting assay we have verified in vitro G4-formation in the HTT elements of D. melanogaster. Namely 3 in Het-A, 8 in TART and 2 in TAHRE. All the G4s are asymmetrically distributed as in canonical telomeres. Bioinformatic analysis showed that asymmetric distribution of potential quadruplex sequences (PQS) is common in telomeric retrotransposons in other Drosophila species. Most of the PQS are located in the gag gene where PQS density correlates with higher DNA sequence conservation and codon selection favoring G4-forming potential. The importance of G4s in non-canonical telomeres is further supported by analysis of telomere-associated retrotransposons from various eukaryotic species including green algae, Diplomonadida, fungi, insects and vertebrates. Virtually all analyzed telomere-associated retrotransposons contained PQS, frequently with asymmetric strand distribution. Comparison with non-telomeric elements showed independent selection of PQS-rich elements from four distinct LINE clades. CONCLUSION: Our findings of strand-biased G4-forming motifs in telomere-associated retrotransposons from various eukaryotic species support the G4-formation as one of the prerequisites for the recruitment of specific retrotransposons to chromosome ends and call for further experimental studies.

• Klíčová slova
• Drosophila, G-quadruplex, Het-A, Retrotransposon, TAHRE, TART, Telomere,
• Publikační typ
• časopisecké články MeSH

Telomere-binding proteins are required for forming the functional structure of chromosome ends and regulating telomerase action. Although a number of candidate proteins have been identified by homology searches to plant genome databases and tested for their affinity to telomeric DNA sequences in vitro, there are minimal data relevant to their telomeric function. To address this problem, we made a collection of cDNAs of putative telomere-binding proteins of Arabidopsis thaliana to analyse their protein-protein interactions with the yeast two-hybrid system. Our results show that one myb-like protein, AtTRP1, interacts specifically with AtKu70, the latter protein having a previously described role in plant telomere metabolism. In analogy to the interaction between human Ku70 and TRF2 proteins, our results suggest that AtTRP1 is a likely homolog of TRF2. The AtTRP1 domain responsible for AtKu70 interaction occurs between amino acid sequence positions 80 and 269. The protein AtTRB1, a member of the single myb histone (Smh) family, shows self-interaction and interactions to the Smh family proteins AtTRB2 and AtTRB3. Protein AtTRB1 also interacts with AtPot1, the Arabidopsis homolog of oligonucleotide-binding-fold-containing proteins which bind G-rich telomeric DNA. In humans, the TRF1-complex recruits hPot1 to telomeres by protein-protein interactions where it is involved in telomere length regulation. Possibly, AtTRB1 has a similar role in recruiting AtPot1.

• MeSH
• Arabidopsis metabolismus   MeSH
• autoradiografie MeSH
• DNA rostlinná chemie   genetika   metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• genetická transkripce MeSH
• klonování DNA MeSH
• methionin chemie   MeSH
• otevřené čtecí rámce MeSH
• posttranslační úpravy proteinů MeSH
• precipitinové testy MeSH
• protein TRF2 chemie   genetika   metabolismus   MeSH
• proteiny huseníčku chemie   genetika   metabolismus   MeSH
• proteiny vázající telomery metabolismus   MeSH
• radioizotopy síry MeSH
• rostlinné proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční delece MeSH
• techniky dvojhybridového systému MeSH
• telomery * MeSH
• terciární struktura proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• methionin MeSH
• protein TRF2 MeSH
• proteiny huseníčku MeSH
• proteiny vázající telomery MeSH
• radioizotopy síry MeSH
• rostlinné proteiny MeSH

BACKGROUND: Unmet medical needs remain in patients with red blood cell transfusion-dependent (RBC-TD) lower-risk myelodysplastic syndromes (LR-MDS) who are not responding to or are ineligible for erythropoiesis-stimulating agents (ESAs). Imetelstat, a competitive telomerase inhibitor, showed promising results in a phase 2 trial. We aimed to compare the RBC transfusion independence (RBC-TI) rate with imetelstat versus placebo in patients with RBC-TD LR-MDS. METHODS: In phase 3 of IMerge, a double-blind, placebo-controlled trial conducted in 118 sites including university hospitals, cancer centres, and outpatient clinics in 17 countries, patients (aged ≥18 years) with ESA-relapsed, ESA-refractory, or ESA-ineligible LR-MDS (low or intermediate-1 risk disease as per International Prognostic Scoring System [IPSS] criteria) were randomly assigned via a computer-generated schedule (2:1) to receive imetelstat 7·5 mg/kg or placebo, administered as a 2-h intravenous infusion, every 4 weeks until disease progression, unacceptable toxic effects, or withdrawal of consent. Randomisation was stratified by previous RBC transfusion burden and IPSS risk group. Patients, investigators, and those analysing the data were masked to group assignment. The primary endpoint was 8-week RBC-TI, defined as the proportion of patients without RBC transfusions for at least 8 consecutive weeks starting on the day of randomisation until subsequent anti-cancer therapy, if any. Primary efficacy analyses were performed in the intention-to-treat population, and safety analyses were conducted in patients who received at least one dose of trial medication or placebo. This trial is registered with ClinicalTrials.gov (NCT02598661; substudy active and recruiting). FINDINGS: Between Sept 11, 2019, and Oct 13, 2021, 178 patients were enrolled and randomly assigned (118 to imetelstat and 60 to placebo). 111 (62%) were male and 67 (38%) were female. 91 (77%) of 118 patients had discontinued treatment by data cutoff in the imetelstat group versus 45 (75%) in the placebo group; a further one patient in the placebo group did not receive treatment. Median follow-up was 19·5 months (IQR 12·0-23·4) in the imetelstat group and 17·5 months (12·1-22·7) in the placebo group. In the imetelstat group, 47 (40% [95% CI 30·9-49·3]) patients had an RBC-TI of at least 8 weeks versus nine (15% [7·1-26·6]) in the placebo group (rate difference 25% [9·9 to 36·9]; p=0·0008). Overall, 107 (91%) of 118 patients receiving imetelstat and 28 (47%) of 59 patients receiving placebo had grade 3-4 treatment-emergent adverse events. The most common treatment-emergent grade 3-4 adverse events in patients taking imetelstat were neutropenia (80 [68%] patients who received imetelstat vs two [3%] who received placebo) and thrombocytopenia (73 [62%] vs five [8%]). No treatment-related deaths were reported. INTERPRETATION: Imetelstat offers a novel mechanism of action with durable transfusion independence (approximately 1 year) and disease-modifying activity for heavily transfused patients with LR-MDS who are not responding to or are ineligible for ESAs. FUNDING: Janssen Research & Development before April 18, 2019, and Geron Corporation thereafter.

• MeSH
• dospělí MeSH
• dvojitá slepá metoda MeSH
• erytropoéza MeSH
• lidé MeSH
• mladiství MeSH
• myelodysplastické syndromy * farmakoterapie   MeSH
• oligonukleotidy * MeSH
• protokoly antitumorózní kombinované chemoterapie MeSH
• trombocytopenie * farmakoterapie   MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky, fáze III MeSH
• práce podpořená grantem MeSH
• randomizované kontrolované studie MeSH
• Názvy látek
• imetelstat MeSH Prohlížeč
• oligonukleotidy * MeSH