scholarly journals Stem-Loop IV of Tetrahymena Telomerase RNA Stimulates Processivity in trans

2003 ◽  
Vol 23 (16) ◽  
pp. 5606-5613 ◽  
Author(s):  
Douglas X. Mason ◽  
Elizabeth Goneska ◽  
Carol W. Greider
Keyword(s):  
Telomerase Activity ◽  
Telomerase Rna ◽  
In Vitro Activity ◽  
Wild Type ◽  
Protein Subunit ◽  
Stem Loop ◽  
Cell Growth Arrest ◽  
In Trans ◽  
Template Region

ABSTRACT Telomerase is a ribonucleoprotein enzyme responsible for the addition of telomeres onto the ends of chromosomes. Short or dysfunctional telomeres can lead to cell growth arrest, apoptosis, and genomic instability. Telomerase uses its RNA subunit to copy a short template region for telomere synthesis. To probe for regions of Tetrahymena telomerase RNA essential for function, we assayed 27 circularly permuted RNA deletions for telomerase in vitro activity and binding to the telomerase reverse transcriptase catalytic protein subunit. We found that stem-loop IV is required for wild-type telomerase activity in vitro and will stimulate processivity when added in trans.

scholarly journals The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans

2019 ◽  
Vol 116 (49) ◽  
pp. 24542-24550 ◽  
Author(s):  
Jiarui Song ◽  
Dhenugen Logeswaran ◽  
Claudia Castillo-González ◽  
Yang Li ◽  
Sreyashree Bose ◽  
...  
Keyword(s):  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Telomerase Rna ◽  
Telomeric Dna ◽  
Structural Element ◽  
Stem Loop ◽  
Evolutionary Pathway ◽  
Pol Iii

Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3′ long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.

scholarly journals Structural features of mouse telomerase RNA are responsible for the lower activity of mouse telomerase versus human telomerase

2006 ◽  
Vol 397 (3) ◽  
pp. 399-406 ◽  
Author(s):  
Scott J. Garforth ◽  
Yan Yun Wu ◽  
Vinayaka R. Prasad
Keyword(s):  
Telomerase Activity ◽  
Structural Features ◽  
In Vitro Translation ◽  
Telomerase Rna ◽  
Level Of Activity ◽  
Human Telomerase ◽  
High Degree ◽  
Made In ◽  
Template Region

Human and mouse telomerases show a high degree of similarity in both the protein and RNA components. Human telomerase is more active and more processive than the mouse telomerase. There are two key differences between hTR [human TR (telomerase RNA)] and mTR (mouse TR) structures. First, the mouse telomerase contains only 2 nt upstream of its template region, whereas the human telomerase contains 45 nt. Secondly, the template region of human telomerase contains a 5-nt alignment domain, whereas that of mouse has only 2 nt. We hypothesize that these differences are responsible for the differential telomerase activities. Mutations were made in both the hTR and mTR, changing the template length and the length of the RNA upstream of the template, and telomerase was reconstituted in vitro using mouse telomerase reverse transcriptase generated by in vitro translation. We show that the sequences upstream of the template region, with a potential to form a double-stranded helix (the P1 helix) as in hTR, increase telomerase activity. The longer alignment domain increases telomerase activity only in the context of the P1 helix. Thus the TR contributes to regulating the level of activity of mammalian telomerases.

scholarly journals A 4-base pair core-enclosing helix in telomerase RNA is essential and binds to the TERT catalytic protein subunit

2020 ◽  
Author(s):  
Melissa A. Mefford ◽  
Evan P. Hass ◽  
David C. Zappulla
Keyword(s):  
Telomerase Activity ◽  
Genome Replication ◽  
Telomerase Rna ◽  
Protein Subunit ◽  
Base Pairs ◽  
Binding Interaction ◽  
Catalytic Core ◽  
Yeast Telomerase

ABSTRACTThe telomerase RNP counters the chromosome end-replication problem, completing genome replication to prevent cellular senescence in yeast, humans, and most other eukaryotes. The telomerase RNP core enzyme is composed of a dedicated RNA subunit and a reverse transcriptase (TERT). Although the majority of the 1157-nt Saccharomyces cerevisiae telomerase RNA, TLC1, is rapidly evolving, the central catalytic core is largely conserved, containing the template, template-boundary helix, pseudoknot, and core-enclosing helix (CEH). Here, we show that 4-base pairs of core-enclosing helix is required for telomerase to be active in vitro and to maintain yeast telomeres in vivo, whereas ΔCEH, 1-bp, and 2-bp alleles do not support telomerase function. Using the CRISPR/dCas9-based “CARRY two-hybrid” assay to assess binding of our CEH mutant RNAs to TERT, we find that the 4-bp CEH RNA binds to TERT, but the shorter-CEH constructs do not, consistent with the telomerase activity and in vivo complementation results. Thus, the CEH is essential in yeast telomerase RNA because it is needed to bind TERT to form the core RNP enzyme. Although the 8 nucleotides that form this 4-bp stem at the base of the CEH are nearly invariant among Saccharomyces species, our results with sequence-randomized and truncated-CEH helices strongly suggest that this binding interaction with TERT is dictated more by secondary than primary structure. In summary, we have mapped an essential binding site in telomerase RNA for TERT that is crucial to form the catalytic core of this biomedically important RNP enzyme.

scholarly journals Folding heterogeneity in the essential human telomerase RNA three-way junction

2019 ◽  
Author(s):  
Christina Palka ◽  
Nicholas M. Forino ◽  
Jendrik Hentschel ◽  
Rhiju Das ◽  
Michael D. Stone
Keyword(s):  
Single Molecule ◽  
Structural Engineering ◽  
De Novo ◽  
Telomerase Rna ◽  
Dna Repeats ◽  
Chemical Mapping ◽  
Protein Subunit ◽  
Stem Loop ◽  
Rnp Complex

AbstractTelomeres safeguard the genome by suppressing illicit DNA damage responses at chromosome termini. In order to compensate for incomplete DNA replication at telomeres, most continually dividing cells, including many cancers, express the telomerase ribonucleoprotein (RNP) complex. Telomerase maintains telomere length by catalyzing de novo synthesis of short DNA repeats using an internal telomerase RNA (TR) template. TRs from diverse species harbor structurally conserved domains that contribute to RNP biogenesis and function. In vertebrate TRs, the conserved regions 4 and 5 (CR4/5) fold into a three-way junction (3WJ) that binds directly to the telomerase catalytic protein subunit and is required for telomerase function. We have analyzed the structural properties of the human TR (hTR) CR4/5 domain using a combination of in vitro chemical mapping, endogenous RNP assembly assays, and single-molecule structural analysis. Our data suggest that a functionally essential stem loop within CR4/5 is not stably folded in the absence of the telomerase reverse transcriptase protein subunit in vitro. Rather, the hTR CR4/5 domain adopts a heterogeneous ensemble of conformations. RNA structural engineering intended to bias the folding landscape of the hTR CR4/5 demonstrates that a stably folded 3WJ motif is necessary but not sufficient to promote assembly of a functional RNP complex. Finally, single-molecule measurements on the hTR CR4/5 domain show that RNP assembly selects for a conformation that is not the major population in the heterogeneous free RNA ensemble, suggesting that non-canonical hTR folds may be required during telomerase biogenesis.

scholarly journals In Vitro Antileishmanial Activity of Nicotinamide

2005 ◽  
Vol 49 (2) ◽  
pp. 808-812 ◽  
Author(s):  
D. Sereno ◽  
A. Monte Alegre ◽  
R. Silvestre ◽  
B. Vergnes ◽  
A. Ouaissi
Keyword(s):  
Leishmania Major ◽  
Growth Arrest ◽  
Antileishmanial Activity ◽  
Vitamin B ◽  
Wild Type ◽  
Intracellular Growth ◽  
Cell Growth Arrest ◽  
Transgenic Parasites

ABSTRACT Our study represents the first report demonstrating the antileishmanial activity of nicotinamide (NAm), a form of vitamin B3. A 5 mM concentration of NAm significantly inhibited the intracellular growth of Leishmania amastigotes and the NAD-dependent deacetylase activity carried by parasites overexpressing Leishmania major SIR2 (LmSIR2). However, the transgenic parasites were as susceptible as the wild-type parasites to NAm-induced cell growth arrest. Therefore, we conclude that NAm inhibits leishmanial growth and that overexpression of LmSIR2 does not overcome this inhibition. The mechanism of the inhibition is not defined but may include other in vivo targets. NAm may thus represent a new antileishmanial agent which could potentially be used in combination with other drugs during therapy.

scholarly journals Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease

2001 ◽  
Vol 82 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Andres Merits ◽  
Lidia Vasiljeva ◽  
Tero Ahola ◽  
Leevi Kääriäinen ◽  
Petri Auvinen
Keyword(s):  
Mammalian Cells ◽  
Active Sites ◽  
Insect Cells ◽  
Semliki Forest Virus ◽  
Point Mutations ◽  
Proteolytic Processing ◽  
Wild Type ◽  
In Trans ◽  
Polyprotein Precursor

The RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C478A, (P2CA) completely abolished the processing of P12CA34, P12CA3 and P2CA3. Co-expression of P23 and P12CA34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D6A, (P1234DA) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.

scholarly journals Efficacy of BB-83698, a Novel Peptide Deformylase Inhibitor, in a Mouse Model of Pneumococcal Pneumonia

2004 ◽  
Vol 48 (1) ◽  
pp. 80-85 ◽  
Author(s):  
E. Azoulay-Dupuis ◽  
J. Mohler ◽  
J. P. Bédos
Keyword(s):  
Mouse Model ◽  
Lung Tissue ◽  
Resistant Strain ◽  
Peptide Deformylase ◽  
In Vitro Activity ◽  
Wild Type ◽  
Virulent Strains ◽  
Resistant Strains

ABSTRACT The efficacy of BB-83698, a novel potent peptide deformylase inhibitor, was evaluated in a mouse model of acute pneumonia. The Streptococcus pneumoniae isolates tested included four virulent strains (one penicillin-susceptible wild-type strain, one macrolide-resistant strain, and two quinolone-resistant mutants [a mutant carrying mutations in ParC and GyrA and an efflux mutant] isogenic to the wild type) and two poorly virulent penicillin-resistant strains. Pneumonia was induced by intratracheal inoculation of 105 CFU (virulent strains) into immunocompetent mice or 107 CFU (less virulent strains) into leukopenic mice. Animals received three or six subcutaneous injections of antibiotics at 12- or 24-h intervals, with antibiotic treatment initiated at 3, 6, 12, or 18 h postinfection (p.i.). BB-83698 showed potent in vitro activity against all strains (MICs, 0.06 to 0.25 μg/ml). In the in vivo model, all control animals died within 2 to 5 days of infection. BB-83698 (80 mg/kg of body weight twice daily or 160 mg/kg once daily) protected 70 to 100% of the animals, as measured 10 days p.i., regardless of the preexisting resistance mechanisms. In contrast, the survival rates for animals treated with the comparator antibiotics were 30% for animals treated with erythromycin (100 mg/kg) and infected with the macrolide-resistant strain, 34% for animals treated with amoxicillin (200 mg/kg every 8 h) and infected with the penicillin-resistant strain, and 0 and 78% for animals treated with ciprofloxacin (250 mg/kg) and infected with the ParC and GyrA mutant and the efflux mutant, respectively. At 80 mg/kg, BB-83698 generated a peak concentration in lung tissue of 61.9 μg/ml within 1 h and areas under the concentration-times curves of 57.4 and 229.4 μg · h/ml for plasma and lung tissue, respectively. The emergence of S. pneumoniae isolates with reduced susceptibilities to BB-83698 was not observed following treatment with a suboptimal dosing regimen. In conclusion, the potent in vitro activity of BB-83698 against S. pneumoniae, including resistant strains, translates into good in vivo efficacy in a mouse pneumonia model.

scholarly journals Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

2008 ◽  
Vol 28 (7) ◽  
pp. 2380-2390 ◽  
Author(s):  
Hong Ji ◽  
Christopher J. Adkins ◽  
Bethany R. Cartwright ◽  
Katherine L. Friedman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Specific Binding ◽  
Telomerase Activity ◽  
Negative Regulator ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomere Length Homeostasis

ABSTRACT In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.

Mouse prostate tumor inhibition via disruption of murine telomerase: In vivo and in vitro data for a new preclinical cancer model

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14631-e14631
Author(s):  
T. Xu ◽  
Y. Xu ◽  
R. Lao ◽  
K. He ◽  
L. Xue ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Cancer Cells ◽  
Cancer Cell Line ◽  
Telomerase Activity ◽  
Telomerase Rna ◽  
Prostate Cancer Cells ◽  
Mouse Prostate

e14631 Background: Telomerase-interference (TI), a novel therapeutic strategy, exploits the high telomerase activity in prostate cancer by introducing a mutated telomerase RNA (MT-Ter) that encodes toxic telomeres. Until now, TI has been tested by targeting human telomerase in tumor cells xenografted into immuno-deficient mice, an inadequate model for predicting efficacy and toxicity. We designed and validated 2 new TI gene constructs that specifically target murine telomerase RNA (mTER), enabling the study of TI in preclinical mouse models that are immuno-competent and that develop endogenous prostate tumors. Methods: We designed 2 constructs and cloned them into a lentiviral delivery system: MT-mTER and siRNA against wild type mTer (α-mTer-siRNA). Using a mouse prostate cancer cell line, E4, we tested the 2 constructs for expression (RT-PCR), telomerase activity (TRAP), and biologic activity (53bp1 DNA damage staining, MTS growth assay, TUNEL and caspase apoptosis assays), as well as in vivo efficacy (NOD-SCID allografts). Results: We confirmed MT-mTER expression (∼50-fold) and showed that α-mTer-siRNA specifically depleted WT-mTER (80% reduction) but not MT-mTER when the 2 constructs are co-expressed; thus, the 2 constructs in combination effectively substituted MT-mTer for WT-mTer in the mouse prostate cancer cells. MT-mTER caused mutant telomeric repeats (TTTGGG instead of TTAGGG) to be added to the ends of telomeres, resulting in rapid telomeric uncapping marked by 53bp1 DNA damage foci (an average 7.5 foci/cell vs. 1.4 foci/cell in vector control). This, in turn, led to rapid and significant apoptosis (>90% TUNEL and caspase +) and growth inhibition in vitro (90% reduction by MTS) and in vivo (75% reduction in tumor allograft size). Conclusions: We successfully designed and validated MT-mTer and α-mTer-siRNA, 2 novel gene constructs that specifically target and co-opt murine telomerase activity within mouse prostate cancer cells. These constructs offer a significant advantage, as they can be used to investigate TI in immuno-competent mice that develop prostate cancer, thereby modeling actual human disease and testing TI-based therapies in a much more informative and authentic manner. No significant financial relationships to disclose.

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