Regulation of Cellular Immortalization and Steady-State Levels of the Telomerase Reverse Transcriptase through Its Carboxy-Terminal Domain

Abstract

Telomerase maintains cell viability and chromosomal stability through the addition of telomere repeats to chromosome ends. The reactivation of telomerase through the upregulation of TERT, the telomerase protein subunit, is an important step during cancer development, yet TERT protein function and regulation remain incompletely understood. Despite its close sequence similarity to human TERT (hTERT), we find that mouse TERT (mTERT) does not immortalize primary human fibroblasts. Here we exploit these differences in activity to understand TERT protein function by creating chimeric mouse-human TERT proteins. Through the analysis of these chimeric TERT proteins, we find that sequences in the human carboxy-terminal domain are critical for telomere maintenance in human fibroblasts. The substitution of the human carboxy-terminal sequences into the mouse TERT protein is sufficient to confer immortalization and maintenance of telomere length and function. Strikingly, we find that hTERT protein accumulates to markedly higher levels than does mTERT protein and that the sequences governing this difference in protein regulation also reside in the carboxyterminal domain. These elevated protein levels, which are characteristic of hTERT, are necessary but not sufficient for telomere maintenance because stabilized mTERT mutants cannot immortalize human cells. Thus, the TERT carboxy terminus contains sequences that regulate TERT protein levels and determinants that are required for productive action on telomere ends.

Telomeric function of mammalian telomerases at short telomeres

“…1A). These results are supported by the observation that the activity reconstituted by the expression of mTERT in primary human BJ fibroblasts stably expressing the SV40 early region is lower than that of hTERT-transduced cells (Middleman et al, 2006).…”

“…The response to telomerase inhibition can vary between human and mouse cells (Marie-Egyptienne et al, 2008;Sachsinger et al, 2001). Species-specific differences in cellular immortalization are also evident because, in the context of fulllength hTERT or mTERT, the hTERT CTE but not the mTERT CTE can promote the immortalization of primary human cells expressing the SV40 early region (Middleman et al, 2006).…”

“…Differences in processivity and the incompatibility of human and mouse telomerases have been mapped to the RNA component (Chen and Greider, 2003;Garforth et al, 2006). However, the contribution of the TERT component to species-specific differences in enzyme activity and processivity is not well understood (Middleman et al, 2006). The response to telomerase inhibition can vary between human and mouse cells (Marie-Egyptienne et al, 2008;Sachsinger et al, 2001).…”

TERT promotes cellular and organismal survival independently of telomerase activity

“…For example, the ectopic expression of mTERT restores telomerase activity in normal human cells, but cannot elongate telomeres (Middleman et al, 2006). Moreover, telomerase activity cannot be reconstituted with hTERT and mTERC in vitro even though mTERC efficiently binds to hTERT Telomerase activity-independent, TERT-mediated protection J Lee et al in vitro (Autexier et al, 1996;Beattie et al, 1998;Chen and Greider, 2003).…”

The human telomerase catalytic subunit and viral telomerase RNA reconstitute a functional telomerase complex in a cell-free system, but not in human cells

“…Conversely, mTERT cannot reconstitute or weakly reconstitutes telomerase activity in pig, chicken and human cells [25][26][27]. mTERT cannot promote the immortalization of primary human cells expressing the SV40 early region, nor can mTERT elongate the shortest telomeres in human cancer cells with alternative lengthening of telomeres (ALT) [25,26].…”