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

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Telomerase is crucial for maintaining telomeres, enabling stem cell immortality and cancer cell proliferation.
• Dysregulation of telomere maintenance leads to stem cell failure syndromes and tumorigenesis.
• Understanding telomerase function at low endogenous levels is vital for both regenerative medicine and cancer research.

Purpose of the Study:

• To investigate the impact of N-terminal telomerase reverse transcriptase fusions on telomerase function in human pluripotent stem cells (hPSCs).
• To analyze telomerase activity and expression at low endogenous levels.
• To elucidate the structural requirements of telomerase for its in vivo function.

Main Methods:

• Genetic engineering of hPSCs to express N-terminal fusion proteins of telomerase reverse transcriptase from the endogenous locus.
• Analysis of telomere length in modified hPSCs and cancer cells.
• Biochemical assays to assess telomerase expression and activity.

Main Results:

• Engineered N-terminal fusions perturbed telomerase function in hPSCs and cancer cells.
• Telomere length defects were observed in modified cells.
• Biochemical analyses indicated multilevel defects in telomerase expression and activity.

Conclusions:

• Small perturbations in telomerase structure can significantly impact its function and telomere maintenance.
• Telomerase function in vivo is highly sensitive to structural modifications.
• This study highlights previously unrecognized complexities in telomerase structural requirements for expression and activity.