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

Abstract

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.

Dynamics of human telomerase recruitment depend on template-telomere base pairing

“…Furthermore, the HaloTag did not affect the functional interaction of telomerase with the telomeric protein TPP1, demonstrated by the increase in processivity in the presence of the shelterin component POT1/TPP1 ( Figure 1, G and H ). We conclude that the introduction of the HaloTag reduces telomerase processivity, as previously shown for Halo- and 3xFLAG-TERT ( Chiba et al. , 2016 ; Schmidt et al.…”

Dynamics of human telomerase recruitment depend on template-telomere base-pairing

“…1G,H). We conclude that the introduction of the HaloTag reduces telomerase processivity, as previously shown for Halo-and 3xFLAG-TERT (Chiba et al, 2016;Schmidt et al, 2016). Importantly, the HaloTag does not affect telomerase activity, RNP assembly, or the interaction of TERT with its telomeric partner, TPP1.…”

Single-molecule imaging of telomerase RNA reveals a Recruitment – Retention model for telomere elongation

“…The different dynamic properties could be explained by a faster rate of acquisition during imaging of HaLo-hTERT compared to MS2-hTR. Although, we cannot rule out that compromised telomerase activity due to hTERT tagging (Chiba et al, 2017; Schmidt et al, 2018) could have altered the dynamic behavior of the enzyme at telomeres.…”