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Kathleen Collins - One of the best experts on this subject based on the ideXlab platform.
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dna binding determinants and cellular thresholds for human Telomerase repeat addition processivity
The EMBO Journal, 2017Co-Authors: Jane Tam, Kathleen CollinsAbstract:The reverse transcriptase Telomerase adds telomeric repeats to chromosome ends. Purified human Telomerase catalyzes processive repeat synthesis, which could restore the full ~100 nucleotides of (T2AG3)n lost from replicated chromosome ends as a single elongation event. Processivity inhibition is proposed to be a basis of human disease, but the impacts of different levels of processivity on telomere maintenance have not been examined. Here, we delineate side chains in the Telomerase active-site cavity important for repeat addition processivity, determine how they contribute to duplex and single-stranded DNA handling, and test the cellular consequences of partial or complete loss of repeat addition processivity for telomere maintenance. Biochemical findings oblige a new model for DNA and RNA handling dynamics in processive repeat synthesis. Biological analyses implicate repeat addition processivity as essential for Telomerase function. However, telomeres can be maintained by Telomerases with lower than wild-type processivity. Furthermore, Telomerases with low processivity dramatically elongate telomeres when overexpressed. These studies reveal distinct consequences of changes in Telomerase repeat addition processivity and expression level on telomere elongation and length maintenance.
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Telomerase: an RNP enzyme synthesizes DNA.
Cold Spring Harbor perspectives in biology, 2011Co-Authors: Elizabeth H Blackburn, Kathleen CollinsAbstract:Telomerase is a eukaryotic ribonucleoprotein (RNP) whose specialized reverse transcriptase action performs de novo synthesis of one strand of telomeric DNA. The resulting Telomerase-mediated elongation of telomeres, which are the protective end-caps for eukaryotic chromosomes, counterbalances the inevitable attrition from incomplete DNA replication and nuclease action. The Telomerase strategy to maintain telomeres is deeply conserved among eukaryotes, yet the RNA component of Telomerase, which carries the built-in template for telomeric DNA repeat synthesis, has evolutionarily diverse size and sequence. Telomerase shows a distribution of labor between RNA and protein in aspects of the polymerization reaction. This article first describes the underlying conservation of a core set of structural features of Telomerase RNAs important for the fundamental polymerase activity of Telomerase. These include a pseudoknot-plus-template domain and at least one other RNA structural motif separate from the template-containing domain. The principles driving the diversity of Telomerase RNAs are then explored. Much of the diversification of Telomerase RNAs has come from apparent gain-of-function elaborations, through inferred evolutionary acquisitions of various RNA motifs used for Telomerase RNP biogenesis, cellular trafficking of enzyme components, and regulation of Telomerase action at telomeres. Telomerase offers broadly applicable insights into the interplay of protein and RNA functions in the context of an RNP enzyme.
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Telomerase in the human organism
Oncogene, 2002Co-Authors: Kathleen Collins, James R. MitchellAbstract:The intent of this review is to describe what is known and unknown about Telomerase in somatic cells of the human organism. First, we consider the Telomerase enzyme. Human Telomerase ribonucleoproteins undergo at least three stages of cellular biogenesis: accumulation, catalytic activation and recruitment to the telomere. Next, we describe the patterns of Telomerase regulation in the human soma. Telomerase activation in some cell types appears to offset proliferation-dependent telomere shortening, delaying but not defeating the inherent mitotic clock. Finally, we elaborate the connection between Telomerase misregulation and human disease, in the contexts of inappropriate Telomerase activation and Telomerase deficiency. We discuss how our current perspectives on Telomerase function could be applied to improving human health.
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Mammalian telomeres and Telomerase.
Current opinion in cell biology, 2000Co-Authors: Kathleen CollinsAbstract:Abstract New features of mammalian telomeres and Telomerase have been identified. Telomeres form t-loops, which engage the 3′ single-stranded DNA overhang in an interaction with double-stranded telomeric repeats. Mammalian Telomerases contain an RNA H/ACA motif and associated protein(s) shared with the H/ACA family of small nucleolar ribonucleoproteins. Essential roles for Telomerase in the sustained viability of cultured tumor cells and in the normal proliferative capacity of human somatic cells have been demonstrated.
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The reverse transcriptase component of the Tetrahymena Telomerase ribonucleoprotein complex
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Kathleen Collins, Leena GandhiAbstract:Telomerase is a eukaryotic reverse transcriptase that adds simple sequence repeats to chromosome ends by copying a template sequence within the RNA component of the enzyme. We describe here the identification of a Tetrahymena Telomerase protein with reverse transcriptase motifs, p133. This subunit is associated with the previously identified Tetrahymena Telomerase RNA and the Telomerase proteins p80 and p95 in immunoprecipitation assays. Therefore, all four known Tetrahymena Telomerase components are present in a single complex. Expressed in rabbit reticulocyte lysate, recombinant p133 and Telomerase RNA alone catalyze a reverse transcriptase activity with some similarities to and some differences from native Tetrahymena Telomerase. These experiments suggest a complexity of Telomerase structure and function.
Steven E. Artandi - One of the best experts on this subject based on the ideXlab platform.
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Regulation of human Telomerase in homeostasis and disease
Nature Reviews Molecular Cell Biology, 2020Co-Authors: Caitlin M. Roake, Steven E. ArtandiAbstract:Telomere length is maintained by Telomerase, which comprises a reverse transcriptase and a template RNA. Telomerase activity is disrupted in several genetic disorders, but is commonly increased in cancer. Recent studies have uncovered many regulatory mechanisms of Telomerase and how Telomerase upregulation in cancer is achieved. Telomerase is a ribonucleoprotein complex, the catalytic core of which includes the Telomerase reverse transcriptase (TERT) and the non-coding human Telomerase RNA (hTR), which serves as a template for the addition of telomeric repeats to chromosome ends. Telomerase expression is restricted in humans to certain cell types, and Telomerase levels are tightly controlled in normal conditions. Increased levels of Telomerase are found in the vast majority of human cancers, and we have recently begun to understand the mechanisms by which cancer cells increase Telomerase activity. Conversely, germline mutations in Telomerase-relevant genes that decrease Telomerase function cause a range of genetic disorders, including dyskeratosis congenita, idiopathic pulmonary fibrosis and bone marrow failure. In this Review, we discuss the transcriptional regulation of human TERT, hTR processing, assembly of the Telomerase complex, the cellular localization of Telomerase and its recruitment to telomeres, and the regulation of Telomerase activity. We also discuss the disease relevance of each of these steps of Telomerase biogenesis.
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TPP1 OB-Fold Domain Controls Telomere Maintenance by Recruiting Telomerase to Chromosome Ends
Cell, 2012Co-Authors: Franklin L. Zhong, Luis F.z. Batista, Adam Freund, Matthew F. Pech, Andrew S. Venteicher, Steven E. ArtandiAbstract:Summary Telomere synthesis in cancer cells and stem cells involves trafficking of Telomerase to Cajal bodies, and Telomerase is thought to be recruited to telomeres through interactions with telomere-binding proteins. Here, we show that the OB-fold domain of the telomere-binding protein TPP1 recruits Telomerase to telomeres through an association with the Telomerase reverse transcriptase TERT. When tethered away from telomeres and other telomere-binding proteins, the TPP1 OB-fold domain is sufficient to recruit Telomerase to a heterologous chromatin locus. Expression of a minimal TPP1 OB-fold inhibits telomere maintenance by blocking access of Telomerase to its cognate binding site at telomeres. We identify amino acids required for the TPP1-Telomerase interaction, including specific loop residues within the TPP1 OB-fold domain and individual residues within TERT, some of which are mutated in a subset of pulmonary fibrosis patients. These data define a potential interface for Telomerase-TPP1 interaction required for telomere maintenance and implicate defective Telomerase recruitment in Telomerase-related disease.
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disruption of Telomerase trafficking by tcab1 mutation causes dyskeratosis congenita
Genes & Development, 2011Co-Authors: Franklin L. Zhong, Sharon A Savage, Marina Shkreli, Neelam Giri, Lea Jessop, Timothy G Myers, Renee Chen, Blanche P Alter, Steven E. ArtandiAbstract:Dyskeratosis congenita (DC) is a genetic disorder of defective tissue maintenance and cancer predisposition caused by short telomeres and impaired stem cell function. Telomerase mutations are thought to precipitate DC by reducing either the catalytic activity or the overall levels of the Telomerase complex. However, the underlying genetic mutations and the mechanisms of telomere shortening remain unknown for as many as 50% of DC patients, who lack mutations in genes controlling telomere homeostasis. Here, we show that disruption of Telomerase trafficking accounts for unknown cases of DC. We identify DC patients with missense mutations in TCAB1, a Telomerase holoenzyme protein that facilitates trafficking of Telomerase to Cajal bodies. Compound heterozygous mutations in TCAB1 disrupt Telomerase localization to Cajal bodies, resulting in misdirection of Telomerase RNA to nucleoli, which prevents Telomerase from elongating telomeres. Our findings establish Telomerase mislocalization as a novel cause of DC, and suggest that Telomerase trafficking defects may contribute more broadly to the pathogenesis of telomere-related disease.
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a human Telomerase holoenzyme protein required for cajal body localization and telomere synthesis
Science, 2009Co-Authors: Andrew S. Venteicher, Eladio Abreu, Zhaojing Meng, Kelly E Mccann, Rebecca M Terns, Timothy D Veenstra, Michael P Terns, Steven E. ArtandiAbstract:Telomerase is a ribonucleoprotein (RNP) complex that synthesizes telomere repeats in tissue progenitor cells and cancer cells. Active human Telomerase consists of at least three principal subunits, including the Telomerase reverse transcriptase, the Telomerase RNA (TERC), and dyskerin. Here, we identify a holoenzyme subunit, TCAB1 (Telomerase Cajal body protein 1), that is notably enriched in Cajal bodies, nuclear sites of RNP processing that are important for Telomerase function. TCAB1 associates with active Telomerase enzyme, established Telomerase components, and small Cajal body RNAs that are involved in modifying splicing RNAs. Depletion of TCAB1 by using RNA interference prevents TERC from associating with Cajal bodies, disrupts Telomerase-telomere association, and abrogates telomere synthesis by Telomerase. Thus, TCAB1 controls Telomerase trafficking and is required for telomere synthesis in human cancer cells.
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identification of atpases pontin and reptin as Telomerase components essential for holoenzyme assembly
Cell, 2008Co-Authors: Andrew S. Venteicher, Zhaojing Meng, Timothy D Veenstra, Philip J Mason, Steven E. ArtandiAbstract:Telomerase is a multisubunit ribonucleoprotein (RNP) complex that adds telomere repeats to the ends of chromosomes. Three essential Telomerase components have been identified thus far: the Telomerase reverse transcriptase (TERT), the Telomerase RNA component (TERC), and the TERC-binding protein dyskerin. Few other proteins are known to be required for human Telomerase function, limiting our understanding of both Telomerase regulation and mechanisms of Telomerase action. Here, we identify the ATPases pontin and reptin as Telomerase components through affinity purification of TERT from human cells. Pontin interacts directly with both TERT and dyskerin, and the amount of TERT bound to pontin and reptin peaks in S phase, evidence for cell-cycle-dependent regulation of TERT. Depletion of pontin and reptin markedly impairs Telomerase RNP accumulation, indicating an essential role in Telomerase assembly. These findings reveal an unanticipated requirement for additional enzymes in Telomerase biogenesis and suggest alternative approaches for inhibiting Telomerase in cancer.
Elizabeth H Blackburn - One of the best experts on this subject based on the ideXlab platform.
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Telomerase: an RNP enzyme synthesizes DNA.
Cold Spring Harbor perspectives in biology, 2011Co-Authors: Elizabeth H Blackburn, Kathleen CollinsAbstract:Telomerase is a eukaryotic ribonucleoprotein (RNP) whose specialized reverse transcriptase action performs de novo synthesis of one strand of telomeric DNA. The resulting Telomerase-mediated elongation of telomeres, which are the protective end-caps for eukaryotic chromosomes, counterbalances the inevitable attrition from incomplete DNA replication and nuclease action. The Telomerase strategy to maintain telomeres is deeply conserved among eukaryotes, yet the RNA component of Telomerase, which carries the built-in template for telomeric DNA repeat synthesis, has evolutionarily diverse size and sequence. Telomerase shows a distribution of labor between RNA and protein in aspects of the polymerization reaction. This article first describes the underlying conservation of a core set of structural features of Telomerase RNAs important for the fundamental polymerase activity of Telomerase. These include a pseudoknot-plus-template domain and at least one other RNA structural motif separate from the template-containing domain. The principles driving the diversity of Telomerase RNAs are then explored. Much of the diversification of Telomerase RNAs has come from apparent gain-of-function elaborations, through inferred evolutionary acquisitions of various RNA motifs used for Telomerase RNP biogenesis, cellular trafficking of enzyme components, and regulation of Telomerase action at telomeres. Telomerase offers broadly applicable insights into the interplay of protein and RNA functions in the context of an RNP enzyme.
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a universal Telomerase rna core structure includes structured motifs required for binding the Telomerase reverse transcriptase protein
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Jue Lin, Arif Hussain, Mira Abraham, Sivan Pearl, Yehuda Tzfati, Tristram G Parslow, Elizabeth H BlackburnAbstract:Telomerase synthesizes telomeric DNA by copying a short template sequence within its Telomerase RNA component. We delineated nucleotides and base-pairings within a previously mapped central domain of the Saccharomyces cerevisiae Telomerase RNA (TLC1) that are important for Telomerase function and for binding to the Telomerase catalytic protein Est2p. Phylogenetic comparison of Telomerase RNA sequences from several budding yeasts revealed a core structure common to Saccharomyces and Kluyveromyces yeast species. We show that in this structure three conserved sequences interact to provide a binding site for Est2p positioned near the template. These results, combined with previous studies on Telomerase RNAs from other budding yeasts, vertebrates, and ciliates, define a minimal universal core for Telomerase RNAs.
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the Telomerase rna pseudoknot is critical for the stable assembly of a catalytically active ribonucleoprotein
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: David Gilley, Elizabeth H BlackburnAbstract:Telomerase is a ribonucleoprotein reverse transcriptase that synthesizes telomeric DNA. A pseudoknot structure is phylogenetically conserved within the RNA component of Telomerase in all ciliated protozoans examined. Here, we report that disruptions of the pseudoknot base pairing within the Telomerase RNA from Tetrahymena thermophila prevent the stable assembly in vivo of an active Telomerase. Restoring the base-pairing potential of the pseudoknot by compensatory changes restores Telomerase activity to essentially wild-type levels. Therefore, the pseudoknot topology rather than sequence is critical for an active Telomerase. Furthermore, we show that disruption of the pseudoknot prevents the association of the RNA with the reverse transcriptase protein subunit of Telomerase. Thus, we provide an example of a structural motif within the Telomerase RNA that is required for Telomerase function and identify the domain that is required for Telomerase complex formation. Hence, we identify a biological role for a pseudoknot: promoting the stable assembly of a catalytically active ribonucleoprotein.
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identification of kluyveromyces lactis Telomerase discontinuous synthesis along the 30 nucleotide long templating domain
Molecular and Cellular Biology, 1998Co-Authors: Tracy B Fulton, Elizabeth H BlackburnAbstract:Telomeres, the essential protein-DNA elements at the ends of most eukaryotic chromosomes, confer chromosome stability and constitute protective terminal caps for the genetic material of the cell (for a review, see reference 45). Telomeric DNA typically consists of tandem arrays of a precisely repeated 5- to 8-bp sequence (for a review, see reference 17). However, the telomeric repeat units of yeast species have greatly diverged in precision and length (4, 26). The budding yeast Kluyveromyces lactis, with a perfectly repeated 25-bp telomeric repeat unit (27), is one of several budding yeasts with exceptionally large telomeric repeats (26). In Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe, the telomeric sequences are shorter, imprecisely repeated units (5′-TG1–3-3′ and primarily 5′-GGTTACA-3′, respectively [18, 36]). Despite their variability between species, all of these telomeric sequences are specified by the enzyme Telomerase. Telomerase, a ribonucleoprotein (RNP) reverse transcriptase, facilitates the complete biosynthesis and maintenance of telomeres. Telomerase activity, as shown initially for the ciliate Tetrahymena thermophila (12) and subsequently for many other eukaryotes (3, 24, 25, 31, 35, 37, 44), is dependent on an integral Telomerase RNA component (13, 14) which contains a sequence that serves as a template for telomeric DNA synthesis (14, 43). While in T. thermophila the templating domain is 5′-CAACCCCAA-3′, complementary to one and a half telomeric repeats (14), in K. lactis it is a 30-nucleotide (nt) RNA sequence complementary to one and a fifth telomere repeat units (27). Specific mutations within the templating domain of both T. thermophila and S. cerevisiae Telomerase RNA drastically reduce or alter Telomerase activity in vitro and in vivo, suggesting that bases in the template are not simply copied but play crucial roles in active-site functions (8, 9, 33). Active-site functions are also carried by the reverse transcriptase protein component of Telomerase (the hTERT gene product), which has been identified in Euplotes aediculatus, yeasts, and human cells (16, 23, 30, 32), confirming that Telomerase requires both reverse transcriptase protein and RNA components. Unlike the extensive RNA genome copying carried out by the more typical reverse transcriptases of viruses and retroposons, the polymerization activity of Telomerase is restricted to copying the discrete template portion of the Telomerase RNA. In vitro, Telomerase elongates a telomeric DNA primer substrate, which aligns within the templating domain via Watson-Crick base pairing (14). The sequence of the oligonucleotide primer determines the positioning of the 3′ end and therefore the site of initiation. Telomerase then extends the primer by polymerization of one nucleotide at a time along the RNA template to the 5′-end boundary. In T. thermophila and S. cerevisiae, mutating the RNA sequence adjacent to the templating domain allows polymerization to proceed beyond the normal template. Hence, Telomerase RNA structures or interactions outside of the template also appear to prevent polymerization beyond the template boundary (1, 33). It is not known whether during primer elongation a constant length of template RNA-product DNA hybrid is maintained (monotonic polymerization) or if the RNA-DNA duplex builds up, although it has been proposed that T. thermophila Telomerase maintains a minimal 3- to 4-bp hybrid during elongation (21). During in vitro reactions, Telomerases from most organisms catalyze multiple rounds of telomere repeat synthesis, and two modes of synthesis, distributive and translocative, have been distinguished. In the distributive synthesis mode, T. thermophila Telomerase dissociates from its DNA product and then binds a new primer to repeat the cycle (5, 21). Translocative synthesis, catalyzed by Telomerase from T. thermophila, E. aediculatus, Saccharomyces castellii, and human cells, involves repositioning of the 3′ end of the newly elongated primer at the beginning of the template without release of the product (for a review, see reference 11). Such translocation and the resulting processive synthesis of multiple repeats on a single primer in vitro are influenced by interactions of the 5′ end of the primer with an anchor site within a protein and/or RNA component of Telomerase (5, 15, 20). Synthesis of small repeats (such as the 6-nt repeat of T. thermophila) requires minimal relative movement within the Telomerase RNP of the built-in RNA template as it crosses the catalytic site of the TERT protein (41). However, the templating domains of the yeast Telomerase RNAs that have been identified, TLC1 RNA from S. cerevisiae and TER1 RNA from K. lactis, are considerably larger, posing interesting mechanistic challenges to telomere repeat synthesis. TLC1 RNA contains a templating domain maximally 17 nt long (39). An 11-nt portion of this domain has been shown to be copied (33), although it is rarely copied in its entirety in vitro, resulting in a series of incomplete single-round extension products (3, 23, 33, 34). Since the frequent stalling exhibited in vitro produces variable 3′-end sequences, alignment of the partially redundant template sequence at telomeric ends can presumably take place in multiple registers and may underlie the degeneracy of the telomeric repeat sequences in vivo (3, 33). The templating domain of K. lactis TER1 RNA is theoretically 30 nt, longer than any examined to date, and in contrast to the irregular repeats of S. cerevisiae telomeres, the telomeric repeat units in K. lactis are tandem arrays of perfect copies of a 25-bp sequence (27). These features suggest that K. lactis Telomerase faithfully copies its entire template. We predicted that the properties that enable K. lactis Telomerase to combine a precise mode of copying with an exceptionally long template would be important for understanding general mechanistic features of Telomerase action. In particular, we anticipated that analysis of Telomerase polymerization along a lengthy template would allow in-depth dissection of the steps in primer elongation occurring within a round of repeat synthesis. In addition, K. lactis has already been established as a highly informative and experimentally advantageous model system for studies on telomere maintenance and length regulation (19, 27, 28). Here we report the identification and characterization of Telomerase activity from K. lactis. We show that in vitro, K. lactis Telomerase catalyzes no more than a single round of repeat synthesis, remains bound to its elongated DNA products, and stalls at specific positions along the template. Stalled complexes result from position-specific arrest and pausing; both are exacerbated by increased complementarity between DNA product and the RNA template. These observations provide new insights into the mechanism of polymerization by Telomerase and have implications for the in vivo functioning of the enzyme.
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Specific RNA residue interactions required for enzymatic functions of Tetrahymena Telomerase.
Molecular and Cellular Biology, 1996Co-Authors: D Gilley, Elizabeth H BlackburnAbstract:The ribonucleoprotein enzyme Telomerase is a specialized reverse transcriptase that synthesizes telomeric DNA by copying a template sequence within the Telomerase RNA. Here we analyze the actions of Telomerase from Tetrahymena thermophila assembled in vivo with mutated or wild-type Telomerase RNA to define further the roles of particular Telomerase RNA residues involved in essential enzymatic functions: templating, substrate alignment, and promotion of polymerization. Position 49 of the Telomerase RNA defined the 3' templating residue boundary, demonstrating that seven positions, residues 43 to 49, are capable of acting as templating residues. We demonstrate directly that positioning of the primer substrate involves Watson-Crick base pairing between the primer with Telomerase RNA residues. Unexpectedly, formation of a Watson-Crick base pair specifically between the primer DNA and Telomerase RNA residue 50 is critical in promoting primer elongation. In contrast, mutant Telomerase with the cytosine at position 49 mutated to a G exhibited efficient 3' mispair extension. This work provides new evidence for specific primer-Telomerase interactions, as well as base-specific interactions involving the Telomerase RNA, playing roles in essential active-site functions of Telomerase.
Lea Harrington - One of the best experts on this subject based on the ideXlab platform.
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functional multimerization of the human Telomerase reverse transcriptase
Molecular and Cellular Biology, 2001Co-Authors: Tara L Beattie, Murray O. Robinson, Wen Zhou, Lea HarringtonAbstract: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 ...
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polymerization defects within human Telomerase are distinct from Telomerase rna and tep1 binding
Molecular Biology of the Cell, 2000Co-Authors: Tara L Beattie, Murray O. Robinson, Wen Zhou, Lea HarringtonAbstract: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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A mammalian Telomerase-associated protein.
Science (New York N.Y.), 1997Co-Authors: Lea Harrington, Timothy Mcphail, Vernon Mar, Wen Zhou, Rena Oulton, Amgen Est Program, Michael Bass, Isabel Arruda, Murray O. RobinsonAbstract:The Telomerase ribonucleoprotein catalyzes the addition of new telomeres onto chromosome ends. A gene encoding a mammalian Telomerase homolog called TP1 (Telomerase-associated protein 1) was identified and cloned. TP1 exhibited extensive amino acid similarity to the Tetrahymena Telomerase protein p80 and was shown to interact specifically with mammalian Telomerase RNA. Antiserum to TP1 immunoprecipitated Telomerase activity from cell extracts, suggesting that TP1 is associated with Telomerase in vivo. The identification of TP1 suggests that Telomerase-associated proteins are conserved from ciliates to humans.
Leena Gandhi - One of the best experts on this subject based on the ideXlab platform.
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The reverse transcriptase component of the Tetrahymena Telomerase ribonucleoprotein complex
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Kathleen Collins, Leena GandhiAbstract:Telomerase is a eukaryotic reverse transcriptase that adds simple sequence repeats to chromosome ends by copying a template sequence within the RNA component of the enzyme. We describe here the identification of a Tetrahymena Telomerase protein with reverse transcriptase motifs, p133. This subunit is associated with the previously identified Tetrahymena Telomerase RNA and the Telomerase proteins p80 and p95 in immunoprecipitation assays. Therefore, all four known Tetrahymena Telomerase components are present in a single complex. Expressed in rabbit reticulocyte lysate, recombinant p133 and Telomerase RNA alone catalyze a reverse transcriptase activity with some similarities to and some differences from native Tetrahymena Telomerase. These experiments suggest a complexity of Telomerase structure and function.
