An alternate splicing variant of the human telomerase catalytic subunit inhibits telomerase activity
An alternate splicing variant of the human telomerase catalytic subunit inhibits telomerase activity
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DOI：
10.1038/sj.neo.7900113
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发表时间：
2000-09-01
期刊：
影响因子：
4.8
通讯作者：
Shay, JW
中科院分区：
文献类型：
--
作者：
Yi, XM;White, DM;Shay, JW
端粒酶，一种细胞逆转录酶，将端粒重复序列添加到染色体末端。在正常的人体细胞中，端粒酶被抑制，端粒逐渐缩短，导致增殖性衰老。端粒酶（hTERT）cDNA的引入足以产生端粒酶活性并使正常人细胞永生化，这表明端粒酶活性的抑制是转录的。端粒酶转录本已被证明有至少六个可变剪接位点（四个插入位点和两个缺失位点），并含有两个或一个缺失位点的变体存在于发展过程中，并在我们调查的一组癌细胞系。一个缺失（β位点）和所有四个插入导致过早的翻译终止，而另一个缺失（α位点）是36 bp，位于逆转录酶（RT）基序A内，这表明该缺失变体可能是端粒酶显性负抑制剂的候选者。我们已经克隆了三个交替剪接的hTERT变体，它们含有α、β或α和β缺失位点。将这些可变剪接变体沿着空载体和野生型hTERT引入正常人成纤维细胞和几种端粒酶阳性的永生细胞系和肿瘤细胞系中。通过蛋白质印迹证实α位点缺失变体（hTERT α（-））构建体的表达。我们发现，没有三个选择性剪接变异体重建成纤维细胞端粒酶活性。然而，hTERT α（-）抑制端粒酶阳性细胞的端粒酶活性，导致端粒缩短并最终导致细胞死亡。这种交替剪接的显性负性变异可能对理解端粒酶在发育、分化和癌症进展过程中的调节很重要。
Telomerase, a cellular reverse transcriptase, adds telomeric repeats to chromosome ends. In normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to proliferative senescence. Introduction of the telomerase (hTERT) cDNA is sufficient to produce telomerase activity and immortalize normal human cells, suggesting that the repression of telomerase activity is transcriptional. The telomerase transcript has been shown to have at least six alternate splicing sites (four insertion sites and two deletion sites), and variants containing both or either of the deletion sites are present during development and in a panel of cancer cell lines we surveyed. One deletion (beta site) and all four insertions cause premature 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 of telomerase. We have cloned three alternately spliced hTERT variants that contain the alpha, beta or both alpha and beta deletion sites. These alternate splicing variants along with empty vector and wild-type hTERT were introduced into normal human fibroblasts and several telomerase-positive immortal and tumor cell lines. Expression of the alpha site deletion variant (hTERT alpha (-)) construct was confirmed by Western blotting. We found that none of the three alternate splicing variants reconstitutes telomerase activity in fibroblasts. However, hTERT alpha (-) inhibits telomerase activities in telomerase-positive cells, causes telomere shortening and eventually cell death. This alternately spliced dominant-negative variant may be important in understanding telomerase regulation during development, differentiation and in cancer progression.