Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Physiology and Pharmacology

Supervisor

Dr. Dean H. Betts

Abstract

Human telomerase, a ribonucleoprotein complex consisting of a catalytic subunit (hTERT) and a RNA template component (TERC), prevents telomere attrition in human embryonic stem cells (hESCs) providing these cells with prolonged replicative capacity. Interestingly, hTERT has several alternatively spliced isoforms, most of which are catalytically inactive. One alternative splicing event involving a complete loss of exons 7 and 8 results in a catalytically inactive isoform called β-hTERT. The β-hTERT is highly expressed in certain cancer types and hESCs. Full-length hTERT (FL-hTERT) is known to translocate from nuclei to mitochondria when cells are exposed to either chronic (oxygen tension) or acute (hydrogen peroxide) oxidative stress. In this study, I examined the alternative splicing of endogenous hTERT transcript in hESC, intra-cellular localization of FL-hTERT and β-hTERT in hESCs, and the effect of overexpressing recombinant hTERT on hESC mitochondria. Human ESCs (HES2 line) were cultured under 2% oxygen and 3xFLAG tagged FL-hTERT and β-hTERT were induced by doxycycline prior to 100μM H2O2 treatment. My results demonstrate that overexpression of transgenic FL-hTERT resulted in a significant increase in endogenous β-hTERT transcript level. Additionally, doxycycline and H2O2 altered the splicing pattern of hTERT in favor of β-hTERT. I demonstrate that not only the full-length- but also β-hTERT can bind TERC, and that β-hTERT overexpression inhibits telomerase activity in vitro. However, I failed to observe the mitochondrial localization of recombinant FL-hTERT or β-hTERT. Lastly, my results demonstrate the pro-apoptotic role of β-hTERT and the pro-survival effect of FL-hTERT under H2O2-induced oxidative stress.

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