An N-terminal Flag-tag impairs TPP1 regulation of telomerase function

Sandhu, Ranjodh; Wei, Derek; Sharma, Madhav; Xu, Lai

doi:10.1016/j.bbrc.2019.03.050

Cited by 6 publications

(6 citation statements)

References 28 publications

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“…However, telomerase recruited to the telomere by TPP1-L cannot efficiently extend chromosome ends. It was recently shown that an N-terminal FLAG tag on TPP1-S inhibits telomere lengthening, suggesting that bulk at the N terminus of TPP1-S interferes with telomere elongation (Sandhu et al, 2019). However, our ability to partially rescue the TPP1-L telomere-lengthening defect by mutagenesis of three N-terminal arginine residues suggests these basic residues also contribute toward telomerase inhibition.…”

Section: Discussionmentioning

confidence: 99%

Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells

et al. 2019

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SUMMARY Telomerase replicates chromosome ends in germ and somatic stem cells to facilitate their continued proliferation. Telomerase action depends on the telomeric protein TPP1, which recruits telomerase to telomeres and facilitates processive DNA synthesis. Here, we identify separation-of-function long (TPP1-L) and short (TPP1-S) isoforms of TPP1 that appear to be generated from separate transcripts and differ only in 86 amino acids at their N terminus. Although both isoforms retain the ability to recruit telomerase, only TPP1-S facilitates efficient telomere synthesis. We find that TPP1-S is the predominant isoform in somatic cells, and strikingly, TPP1-L is the major isoform in differentiated male germ cells. We observed that TERT expression persists in these germ cells, suggesting that TPP1-L could restrain telomerase in this context. We show how differential expression of TPP1 isoforms determines telomerase function and demonstrate how alternative transcription start sites allow one gene to perform distinct functions in different biological contexts.

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Section: Discussionmentioning

confidence: 99%

Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells

et al. 2019

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“…Flag-TPP1, GFP-TPP1, GFP-POT1, and Flag-TERT lentiviral constructs contain respective cDNA driven by a CMV promoter, followed by an internal ribosome entry site and a hygromycin resistance gene. For full-length TPP1 expression, TPP1 coding sequence from amino acids 87 to 544, as previously defined (24,41), was used. Telomerase RNA (hTR) expression was driven by an IU1 promoter (42).…”

Section: Methodsmentioning

confidence: 99%

“…CRISPR-Cas9-mediated genome editing, screening of knock-in clones, direct telomerase activity assay, TRF analysis, and TRAP telomerase activity assay were performed using published protocols (24,32,41). See SI Appendix, SI Materials and Methods for full details.…”

Section: Methodsmentioning

confidence: 99%

The structurally conserved TELR region on shelterin protein TPP1 is essential for telomerase processivity but not recruitment

Sandhu

Sharma

Wei

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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The shelterin protein TPP1 is involved in both recruiting telomerase and stimulating telomerase processivity in human cells. Assessing the in vivo significance of the latter role of TPP1 has been difficult, because TPP1 mutations that perturb telomerase function tend to abolish both telomerase recruitment and processivity. The Saccharomyces cerevisiae telomerase-associated Est3 protein adopts a protein fold similar to the N-terminal region of TPP1. Interestingly, a previous structure-guided mutagenesis study of Est3 revealed a TELR surface region that regulates telomerase function via an unknown mechanism without affecting the interaction between Est3 and telomerase [T. Rao et al., Proc. Natl. Acad. Sci. U.S.A. 111, 214–218 (2014)]. Here, we show that mutations within the structurally conserved TELR region on human TPP1 impaired telomerase processivity while leaving telomerase recruitment unperturbed, hence uncoupling the two roles of TPP1 in regulating telomerase. Telomeres in cell lines containing homozygous TELR mutations progressively shortened to a critical length that caused cellular senescence, despite the presence of abundant telomerase in these cells. Our findings not only demonstrate that telomerase processivity can be regulated by TPP1 in a process separable from its role in recruiting telomerase, but also establish that the in vivo stimulation of telomerase processivity by TPP1 is critical for telomere length homeostasis and long-term viability of human cells.

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“…The FLAG tag was originally designed as a hydrophilic tag which could be removed from the target protein by enterokinases (Sasaki et al 2012). FLAG tags were added to the N and C-terminal of Smp24 to determine the effect on function as N-terminal tags have previously been shown to inhibit the functionality of proteins (Sandhu et al 2019).…”

Section: Discussionmentioning

confidence: 99%

“…The C-terminally tagged peptide was ultimately chosen for protein overproduction due to a higher level of expression as determined by dot blot analysis when compared with the N-terminally tagged protein. The sensitivity of the commercially available antibodies is not always sufficient to detect low-level protein expression (Sandhu et al 2019), which could explain the lack of detection for the N-terminally tagged protein within the culture supernatant. It is also possible the N-terminally tagged protein did not express, though the reason for this is unknown.…”

Section: Discussionmentioning

confidence: 99%

Antimicrobial Peptides from Venom: Structure, Function and Toxicity

Rawson¹

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An N-terminal Flag-tag impairs TPP1 regulation of telomerase function

Cited by 6 publications

References 28 publications

Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells

Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells

The structurally conserved TELR region on shelterin protein TPP1 is essential for telomerase processivity but not recruitment

Antimicrobial Peptides from Venom: Structure, Function and Toxicity

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