Minimized human telomerase maintains telomeres and resolves endogenous roles of H/ACA proteins, TCAB1, and Cajal bodies

Vogan, Jacob M.; Zhang, Xiaozhu; Youmans, Daniel T.; Regalado, Samuel G.; Johnson, Joshua Z; Hockemeyer, Dirk; Collins, Kathleen

doi:10.7554/elife.18221

Cited by 49 publications

(97 citation statements)

References 51 publications

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“…Human U2OS cells were transiently transfected using calcium phosphate for expression of human TERT N-terminally tagged with tandem Protein A domains and triple FLAG peptide epitope (ZZF-TERT) from a pcDNA3.1 expression construct (13) and the hTRmin from pBS constructs. Cells were harvested after 48 hours and lysed by 3 freeze-thaw cycles in hypotonic lysis buffer (HLB: 20 mM HEPES at pH 8.0, 2 mM MgCl 2 , 0.2 mM EGTA, 10% glycerol, 0.1% NP-40, 1 mM DTT, 0.1 mM PMSF and Sigma protease inhibitor cocktail).…”

Section: Methodsmentioning

confidence: 99%

Mechanisms of template handling and pseudoknot folding in human telomerase and their manipulation to expand the sequence repertoire of processive repeat synthesis

Deshpande

Collins

2018

Nucleic Acids Research

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Telomerase adds telomeric repeats to chromosome ends by processive copying of a template within the telomerase RNA bound to telomerase reverse transcriptase. Telomerase RNAs have single-stranded regions that separate the template from a 5′ stem and 3′ pseudoknot, and mammals gained additional stem P2a.1 separating the template from the pseudoknot. Using human telomerase, we show that the length of template 3′-flanking single-stranded RNA is a determinant of repeat addition processivity whereas template 5′-flanking single-stranded RNA and P2a.1 are critical for activity but not processivity. In comparison, requirements for the template sequence itself are confounding: different substitutions of the same position have strikingly different consequences, from improved processivity and activity to complete inactivation. We discovered that some altered-template sequences stabilize an alternative RNA conformation that precludes the pseudoknot by base-pairing of one pseudoknot strand to the template 3′ end. Using mutations to reduce over-stability of the alternative conformation, we restore high activity and processivity to otherwise inactive altered-template telomerase ribonucleoproteins. In cells, over-stabilization or destabilization of the alternative state severely inhibited biogenesis of active telomerase. Our findings delineate roles for human telomerase RNA template-flanking regions, establish a biologically relevant pseudoknot-alternative RNA conformation, and expand the repertoire of human telomerase repeat synthesis.

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Section: Methodsmentioning

confidence: 99%

Mechanisms of template handling and pseudoknot folding in human telomerase and their manipulation to expand the sequence repertoire of processive repeat synthesis

Deshpande

Collins

2018

Nucleic Acids Research

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“…In fixed cells, fluorescence in situ hybridization (FISH) for examination of the telomerase RNA or the localization of Cajal bodies to telomeres has been used as a proxy for the localization of TERT to telomeres (32,33). However, recent genetic data suggest that these associations might not be directly reporting on telomerase action at telomeres (34)(35)(36). With the advent of genome editing in human pluripotent stem cells (reviewed in reference 37), an experimental system became available that can overcome these challenges.…”

mentioning

confidence: 99%

“…To this end, we knocked out TERT (TERT Ϫ/Ϫ ) in HCT116 cells (36), a colon carcinoma cell line, using the same strategy as previously described in hESCs (18). In these TERT Ϫ/Ϫ cells, telomeres shorten progressively, and cells eventually die due to critically short telomeres (36). If TERT is ectopically expressed in these cells, viability and telomere maintenance are restored and telomeres elongate.…”

Section: Flag-gs20mentioning

confidence: 99%

“…Therefore, we wanted to determine whether the effects that we detected for the N-terminal tagging of TERT at its endogenous locus in hESCs are specific to the stem cell system compared to the field standard transgene-mediated TERT overexpression in cancer cell lines. To this end, we knocked out TERT (TERT Ϫ/Ϫ ) in HCT116 cells (36), a colon carcinoma cell line, using the same strategy as previously described in hESCs (18). In these TERT Ϫ/Ϫ cells, telomeres shorten progressively, and cells eventually die due to critically short telomeres (36).…”

Section: Flag-gs20mentioning

confidence: 99%

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Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo

Chiba

Vogan

et al. 2017

Molecular and Cellular Biology

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Telomerase action at telomeres is essential for the immortal phenotype of stem cells and the aberrant proliferative potential of cancer cells. Insufficient telomere maintenance can cause stem cell and tissue failure syndromes, while increased telomerase levels are associated with tumorigenesis. Both pathologies can arise from only small perturbation of telomerase function. To analyze telomerase at its low endogenous expression level, we genetically engineered human pluripotent stem cells (hPSCs) to express various N-terminal fusion proteins of the telomerase reverse transcriptase from its endogenous locus. Using this approach, we found that these modifications can perturb telomerase function in hPSCs and cancer cells, resulting in telomere length defects. Biochemical analysis suggests that this defect is multileveled, including changes in expression and activity. These findings highlight the unknown complexity of telomerase structural requirements for expression and function in vivo.

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“…In contrast, the higher affinity binding of the human STE (CR4/5 P5-P6-P6.1 three-helix junction) apparently does not require additional proteins, as a minimized hTER with the H/ACA protein binding region deleted assembles an RNP in vivo that maintains stable telomere length homeostasis [48]. …”

Section: Ter Structure and Assembly With Tertmentioning

confidence: 99%

Structural biology of telomerase and its interaction at telomeres

Wang

Feigon

2017

Current Opinion in Structural Biology

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Telomerase is an RNP that synthesizes the 3′ ends of linear chromosomes and is an important regulator of telomere length. It contains a single long non-coding telomerase RNA (TER), telomerase reverse transcriptase (TERT), and other proteins that vary among organisms. Recent progress in structural biology of telomerase includes reports of the first cryo-electron microscopy structure of telomerase, from Tetrahymena, new crystal structures of TERT domains, telomerase RNA structures and models, and identification in Tetrahymena telomerase holoenzyme of human homologues of telomere-associated proteins that have provided a more unified view of telomerase interaction at telomeres as well as insights into the role of telomerase RNA in activity and assembly.

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Minimized human telomerase maintains telomeres and resolves endogenous roles of H/ACA proteins, TCAB1, and Cajal bodies

Cited by 49 publications

References 51 publications

Mechanisms of template handling and pseudoknot folding in human telomerase and their manipulation to expand the sequence repertoire of processive repeat synthesis

Mechanisms of template handling and pseudoknot folding in human telomerase and their manipulation to expand the sequence repertoire of processive repeat synthesis

Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo

Structural biology of telomerase and its interaction at telomeres

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