Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding

The stem terminus element (STE), which was discovered 13 y ago in human telomerase RNA, is required for telomerase activity, yet its mode of action is unknown. We report that the Schizosaccharomyces pombe telomerase RNA, TER1 (telomerase RNA 1), also contains a STE, which is essential for telomere maintenance. Cells expressing a partial loss-of-function TER1 STE allele maintained short stable telomeres by a recombination-independent mechanism. Remarkably, the mutant telomere sequence was different from that of wild-type cells. Generation of the altered sequence is explained by reverse transcription into the template boundary element, demonstrating that the STE helps maintain template boundary element function. The altered telomeres bound less Pot1 (protection of telomeres 1) and Taz1 (telomere-associated in Schizosaccharomyces pombe 1) in vivo. Thus, the S. pombe STE, although distant from the template, ensures proper telomere sequence, which in turn promotes proper assembly of the shelterin complex.

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Identification of Two RNA-binding Proteins Associated with Human Telomerase RNA

Molecular Biology of the Cell ◽

10.1091/mbc.11.3.999 ◽

2000 ◽

Vol 11 (3) ◽

pp. 999-1010 ◽

Cited By ~ 88

Author(s):

Siyuan Le ◽

Rolf Sternglanz ◽

Carol W. Greider

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Telomerase Activity ◽

Telomere Maintenance ◽

Telomerase Rna ◽

Human Telomerase Reverse Transcriptase ◽

Ribonucleoprotein Complexes ◽

Cell Extracts ◽

Human Telomerase

Telomerase plays a crucial role in telomere maintenance in vivo. To understand telomerase regulation, we have been characterizing components of the enzyme. To date several components of the mammalian telomerase holoenzyme have been identified: the essential RNA component (human telomerase RNA [hTR]), the catalytic subunit human telomerase reverse transcriptase (hTERT), and telomerase-associated protein 1. Here we describe the identification of two new proteins that interact with hTR: hStau and L22. Antisera against both proteins immunoprecipitated hTR, hTERT, and telomerase activity from cell extracts, suggesting that the proteins are associated with telomerase. Both proteins localized to the nucleolus and cytoplasm. Although these proteins are associated with telomerase, we found no evidence of their association with each other or with telomerase-associated protein 1. Both hStau and L22 are more abundant than TERT. This, together with their localization, suggests that they may be associated with other ribonucleoprotein complexes in cells. We propose that these two hTR-associated proteins may play a role in hTR processing, telomerase assembly, or localization in vivo.

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SIRT1 contributes to telomere maintenance and augments global homologous recombination

The Journal of Cell Biology ◽

10.1083/jcb.201005160 ◽

2010 ◽

Vol 191 (7) ◽

pp. 1299-1313 ◽

Cited By ~ 156

Author(s):

Jose A. Palacios ◽

Daniel Herranz ◽

Maria Luigia De Bonis ◽

Susana Velasco ◽

Manuel Serrano ◽

...

Keyword(s):

Homologous Recombination ◽

Telomerase Activity ◽

Histone Deacetylation ◽

Telomere Maintenance ◽

Loss Of Function ◽

Entire Genome ◽

Telomere Shortening ◽

Telomere Biology ◽

Sir Complex

Yeast Sir2 deacetylase is a component of the silent information regulator (SIR) complex encompassing Sir2/Sir3/Sir4. Sir2 is recruited to telomeres through Rap1, and this complex spreads into subtelomeric DNA via histone deacetylation. However, potential functions at telomeres for SIRT1, the mammalian orthologue of yeast Sir2, are less clear. We studied both loss of function (SIRT1 deficient) and gain of function (SIRT1super) mouse models. Our results indicate that SIRT1 is a positive regulator of telomere length in vivo and attenuates telomere shortening associated with aging, an effect dependent on telomerase activity. Using chromatin immunoprecipitation assays, we find that SIRT1 interacts with telomeric repeats in vivo. In addition, SIRT1 overexpression increases homologous recombination throughout the entire genome, including telomeres, centromeres, and chromosome arms. These findings link SIRT1 to telomere biology and global DNA repair and provide new mechanistic explanations for the known functions of SIRT1 in protection from DNA damage and some age-associated pathologies.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915312116 ◽

2019 ◽

Vol 116 (49) ◽

pp. 24542-24550 ◽

Cited By ~ 8

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.

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Polymerization Defects within Human Telomerase Are Distinct from Telomerase RNA and TEP1 Binding

Molecular Biology of the Cell ◽

10.1091/mbc.11.10.3329 ◽

2000 ◽

Vol 11 (10) ◽

pp. 3329-3340 ◽

Cited By ~ 59

Author(s):

Tara L. Beattie ◽

Wen Zhou ◽

Murray O. Robinson ◽

Lea Harrington

Keyword(s):

Telomerase Activity ◽

Amino Terminus ◽

Telomerase Rna ◽

Carboxy Terminus ◽

Active Core ◽

Human Telomerase ◽

Precise Role

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.

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Disease-Associated Human Telomerase RNA Variants Show Loss of Function for Telomere Synthesis without Dominant-Negative Interference

Molecular and Cellular Biology ◽

10.1128/mcb.00777-08 ◽

2008 ◽

Vol 28 (20) ◽

pp. 6510-6520 ◽

Cited By ~ 16

Author(s):

Timothy M. Errington ◽

Dragony Fu ◽

Judy M. Y. Wong ◽

Kathleen Collins

Keyword(s):

Negative Impact ◽

Dominant Negative ◽

Telomere Maintenance ◽

General Mechanism ◽

Genome Replication ◽

Telomerase Rna ◽

Loss Of Function ◽

Wild Type ◽

Human Telomerase

ABSTRACT Telomerase adds simple-sequence repeats to chromosome ends to offset the terminal sequence loss inherent in each cycle of genome replication. Inherited mutations in genes encoding subunits of the human telomerase holoenzyme give rise to disease phenotypes including hematopoietic failure and pulmonary fibrosis. Disease-associated variants of the human telomerase RNA are expressed in heterozygous combination with wild-type telomerase RNA. Here, we exploit a sensitized human primary cell assay system to investigate the biological function of disease-linked telomerase RNA variants and their impact on the function of coexpressed wild-type telomerase RNA. We find that telomerase RNA variants discovered in patients with dyskeratosis congenita or aplastic anemia show loss of function without any indication of dominant-negative impact on telomere maintenance by the coexpressed wild-type RNA. To reconcile this result with contradictory findings from reconstitution assays in vitro, we demonstrate that the lack of dominant-negative impact on telomere maintenance correlates with physiological assembly of active human telomerase holoenzyme ribonucleoproteins harboring monomers rather than higher-order multimers of telomerase RNA and telomerase reverse transcriptase. These findings support loss of function of telomerase RNA as a general mechanism of human disease.

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Expression of Telomerase RNA Template, but Not Telomerase Reverse Transcriptase, Is Limiting for Telomere Length Maintenance In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.24.16.7024-7031.2004 ◽

2004 ◽

Vol 24 (16) ◽

pp. 7024-7031 ◽

Cited By ~ 76

Author(s):

Y. Jeffrey Chiang ◽

Michael T. Hemann ◽

Karen S. Hathcock ◽

Lino Tessarollo ◽

Lionel Feigenbaum ◽

...

Keyword(s):

Reverse Transcriptase ◽

Telomere Length ◽

Telomerase Activity ◽

Telomere Maintenance ◽

Gene Copy ◽

Telomerase Reverse Transcriptase ◽

Telomerase Rna ◽

Wild Type ◽

Telomere Elongation

ABSTRACT Telomerase consists of two essential components, the telomerase RNA template (TR) and telomerase reverse transcriptase (TERT). The haplo-insufficiency of TR was recently shown to cause one form of human dyskeratosis congenita, an inherited disease marked by abnormal telomere shortening. Consistent with this finding, we recently reported that mice heterozygous for inactivation of mouse TR exhibit a similar haplo-insufficiency and are deficient in the ability to elongate telomeres in vivo. To further assess the genetic regulation of telomerase activity, we have compared the abilities of TR-deficient and TERT-deficient mice to maintain or elongate telomeres in interspecies crosses. Homozygous TERT knockout mice had no telomerase activity and failed to maintain telomere length. In contrast, TERT+/− heterozygotes had no detectable defect in telomere elongation compared to wild-type controls, whereas TR+/− heterozygotes were deficient in telomere elongation. Levels of TERT mRNA in heterozygous mice were one-third to one-half the levels expressed in wild-type mice, similar to the reductions in telomerase RNA observed in TR heterozygotes. These findings indicate that both TR and TERT are essential for telomere maintenance and elongation but that gene copy number and transcriptional regulation of TR, but not TERT, are limiting for telomerase activity under the in vivo conditions analyzed.

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Functional Characterization of Telomerase RNA Variants Found in Patients with Hematological Disorders.

Blood ◽

10.1182/blood.v104.11.2832.2832 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2832-2832

Author(s):

Hinh Ly ◽

Rodrigo T. Calado ◽

Paulette A. Allard ◽

Gabriela M. Baerlocher ◽

Peter M. Lansdorp ◽

...

Keyword(s):

Aplastic Anemia ◽

Bone Marrow Failure ◽

Functional Characterization ◽

Telomerase Activity ◽

Telomere Maintenance ◽

Loss Of Function ◽

Hematological Disorders ◽

Enzymatic Function ◽

Bone Marrow Failure Syndromes ◽

Human Telomerase

Abstract Human telomerase uses a portion of its integral RNA component (hTER) as the template to synthesize telomeres at chromosome ends. hTER sequence polymorphisms have been observed in some patients with bone marrow failure syndromes such as aplastic anemia, but the functional significance of most such variants is unknown. Here, we report the functional characteristics of ten previously-described and two newly discovered hTER disease-associated polymorphisms. Most of these hTER variants adversely affected telomerase enzymatic function as measured in the telomerase reconstituted human cells. Similar loss-of-function effects were also seen directly in primary lymphocytes collected from two of the patients. The majority of the functional deficits stemmed from perturbations of the predicted hTER RNA secondary structure, and corresponded well with the degrees of telomere shortening observed in patients. In contrast, hTER variants anticipated to be inconsequential polymorphisms, which were also found in healthy subjects, did not interfere with telomerase function. Loss of telomerase activity and of telomere maintenance resulting from inherited hTER mutations may predispose some patients to aplastic anemia and other marrow failure disorders.

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Human Telomerase RNA: Telomerase Component or More?

Biomolecules ◽

10.3390/biom10060873 ◽

2020 ◽

Vol 10 (6) ◽

pp. 873 ◽

Cited By ~ 1

Author(s):

Maria Rubtsova ◽

Olga Dontsova

Keyword(s):

Oxidative Stress ◽

Regulation Of Gene Expression ◽

Telomerase Activity ◽

Telomere Maintenance ◽

Telomerase Rna ◽

Biological Processes ◽

Protein Subunit ◽

Independent Components ◽

Cellular Processes ◽

Human Telomerase

Telomerase is a ribonucleoprotein complex that maintains the lengths of telomeres. Most studies of telomerase function have focused on the involvement of telomerase activation in the immortalization of cancer cells and cellular rejuvenation. However, some studies demonstrated that the results do not meet expectations for telomerase action in telomere maintenance. Recent results give reason to think that major telomerase components—the reverse transcriptase protein subunit and telomerase RNA—may participate in many cellular processes, including the regulation of apoptosis and autophagy, cell survival, pro-proliferative effects, regulation of gene expression, and protection against oxidative stress. However, the difficulties faced by scientist when researching telomerase component functions often reduce confidence in the minor effects observed in experiments. In this review, we focus on the analysis of the functions of telomerase components (paying more attention to the telomerase RNA component), both as a complex and as independent components, providing effects that are not associated with telomerase activity and telomere length maintenance. Despite the fact that the data on alternative roles of telomerase components look illusory, it would be wrong to completely reject the possibility of their involvement in other biological processes excluded from research/discussion. Investigations to improve the understanding of every aspect of the functioning of telomerase components will provide the basis for a more precise development of approaches to regulate cellular homeostasis, which is important for carcinogenesis and aging.

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Functional Multimerization of the Human Telomerase Reverse Transcriptase

Molecular and Cellular Biology ◽

10.1128/mcb.21.18.6151-6160.2001 ◽

2001 ◽

Vol 21 (18) ◽

pp. 6151-6160 ◽

Cited By ~ 92

Author(s):

Tara L. Beattie ◽

Wen Zhou ◽

Murray O. Robinson ◽

Lea Harrington

Keyword(s):

Reverse Transcriptase ◽

Telomerase Activity ◽

Telomerase Reverse Transcriptase ◽

Telomerase Rna ◽

Human Telomerase Reverse Transcriptase ◽

Catalytic Core ◽

Cell Extracts ◽

Human Telomerase

ABSTRACT The telomerase enzyme exists as a large complex (∼1,000 kDa) in mammals and at minimum is composed of the telomerase RNA and the catalytic subunit telomerase reverse transcriptase (TERT). In Saccharomyces cerevisiae, telomerase appears to function as an interdependent dimer or multimer in vivo (J. Prescott and E. H. Blackburn, Genes Dev. 11:2790–2800, 1997). However, the requirements for multimerization are not known, and it remained unclear whether telomerase exists as a multimer in other organisms. We show here that human TERT (hTERT) forms a functional multimer in a rabbit reticulocyte lysate reconstitution assay and in human cell extracts. Two separate, catalytically inactive TERT proteins can complement each other in trans to reconstitute catalytic activity. This complementation requires the amino terminus of one hTERT and the reverse transcriptase and C-terminal domains of the second hTERT. The telomerase RNA must associate with only the latter hTERT for reconstitution of telomerase activity to occur. Multimerization of telomerase also facilitates the recognition and elongation of substrates in vitro and in vivo. These data suggest that the catalytic core of human telomerase may exist as a functionally cooperative dimer or multimer in vivo.

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