An alternate splicing variant of the human telomerase catalytic subunit inhibits telomerase activity

Telomerase, a cellular reverse transcriptase, adds telomeric repeats to chr omosome ends. In normal human somatic cells, telomerase is repressed and te lomeres progressively shorten, leading to proliferative senescence. Introdu ction of the telomerase (hTERT) cDNA is sufficient to produce telomerase ac tivity and immortalize normal human cells, suggesting that the repression o f telomerase activity is transcriptional. The telomerase transcript has bee n shown to have at least six alternate splicing sites (four insertion sites and two deletion sites), and variants containing both or either of the del etion sites are present during development and in a panel of cancer cell li nes we surveyed. One deletion (beta site) and all four insertions cause pre mature translation terminations, whereas the other deletion (alpha site) is 36 bp and lies within reverse transcriptase (RT) motif A, suggesting that this deletion variant may be a candidate as a dominant-negative inhibitor o f telomerase. We have cloned three alternately spliced hTERT variants that contain the alpha, beta or both alpha and beta deletion sites. These altern ate splicing variants along with empty vector and wild-type hTERT were intr oduced into normal human fibroblasts and several telomerase-positive immort al and tumor cell lines. Expression of the alpha site deletion variant (hTE RT alpha (-)) construct was confirmed by Western blotting. We found that no ne of the three alternate splicing variants reconstitutes telomerase activi ty in fibroblasts. However, hTERT alpha (-) inhibits telomerase activities in telomerase-positive cells, causes telomere shortening and eventually cel l death. This alternately spliced dominant-negative variant may be importan t in understanding telomerase regulation during development, differentiatio n and in cancer progression.