Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT

Abstract

The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA polymerases cannot fully replicate the ends of linear molecules. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumours. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division. It has been proposed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation. In addition to the human telomerase RNA component (hTR; ref. 11), TR1/TLP1 (refs 12, 13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme, has been identified in humans. More recently, the human telomerase reverse transcriptase (hTRT; refs 15, 16), which is homologous to the reverse transcriptase (RT)-like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosaccharomyces pombe (5pTrt1) telomerases, has been reported to be a telomerase protein subunit. A catalytic function has been demonstrated for Est2p in the RT-like class but not for p80 or its homologues. We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activity that exhibits enzymatic properties like those of the native enzyme. Single amino-acid changes in conserved telomerase-specific and RT motifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase. Normal human diploid cells transiently expressing hTRT possessed telomerase activity, demonstrating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells. The ability to reconstitute telomerase permits further analysis of its biochemical and biological roles in cell aging and carcinogenesis.

Up-regulation of CIR1/CROC1 expression upon cell immortalization and in tumor-derived human cell lines

“…Previously, we had shown that in the pre-immortal HA1 clone, telomere length declines with cell division and telomerase remains inactive until the cells reach proliferative crisis, while in immortal HA1 cells telomere length stabilizes and telomerase is activated (Counter et al, 1992;Avilion et al, 1996). Recently, we also found that up-regulation of the mRNA encoding the catalytic subunit of human telomerase, hTERT (Meyerson et al, 1997;Weinrich et al, 1997;Nakamura and Cech, 1998), coincides with the emergence of immortal HA1 cells (Meyerson et al, 1997).…”

Combination therapy of malignant glioma cells with 2-5A-antisense telomerase RNA and recombinant adenovirus p53

“…37,38 Although hTR and hTERT are essential for telomerase activity, the expression of hTERT is closely associated with telomerase activity. 39,40 Therefore, to inhibit telomerase activity significantly, the hTERT may be more attractive as a target than the hTR. More recently, it has been demonstrated that the complete inhibition of telomerase using a dominant-negative mutant of hTERT resulted in reduction in telomere length and apoptotic death of tumor cells.…”

Expression of mouse telomerase reverse transcriptase during development, differentiation and proliferation

“…To test directly whether the assembled mTERT cDNA encoded catalytic activity, in vitro telomerase reconstitution assays were performed according to Weinrich et al (1997). mTERT was expressed alone or in combination with hTR and telomerase activity was measured by a modi®ed version of the TRAP assay ; for a positive control, parallel assays were performed with hTERT and hTR.…”