Marie-Egyptienne Delphine T, Cerone Maria Antonietta, Londoño-Vallejo J Arturo, Autexier Chantal
Department of Anatomy and Cell Biology, Institut Curie 26 rue d'Ulm, 75248 Paris, CEDEX 05, France.
Nucleic Acids Res. 2005 Sep 28;33(17):5446-57. doi: 10.1093/nar/gki848. Print 2005.
The phylogenetically-derived secondary structures of telomerase RNAs (TR) from ciliates, yeasts and vertebrates are surprisingly conserved and contain a pseudoknot domain at a similar location downstream of the template. As the pseudoknot domains of Tetrahymena TR (tTR) and human TR (hTR) mediate certain similar functions, we hypothesized that they might be functionally interchangeable. We constructed a chimeric TR (htTR) by exchanging the hTR pseudoknot sequences for the tTR pseudoknot region. The chimeric RNA reconstituted human telomerase activity when coexpressed with hTERT in vitro, but exhibited defects in repeat addition processivity and levels of DNA synthesis compared to hTR. Activity was dependent on tTR sequences within the chimeric RNA. htTR interacted with hTERT in vitro and dimerized predominantly via a region of its hTR backbone, the J7b/8a loop. Introduction of htTR in telomerase-negative cells stably expressing hTERT did not reconstitute an active enzyme able to elongate telomeres. Thus, our results indicate that the chimeric RNA reconstituted a weakly active nonprocessive human telomerase enzyme in vitro that was defective in telomere elongation in vivo. This suggests that there may be species-specific requirements for pseudoknot functions.
来自纤毛虫、酵母和脊椎动物的端粒酶RNA（TR）在系统发育上衍生的二级结构惊人地保守，并且在模板下游的相似位置含有一个假结结构域。由于嗜热四膜虫TR（tTR）和人类TR（hTR）的假结结构域介导某些相似的功能，我们推测它们在功能上可能是可互换的。我们通过将hTR假结序列替换为tTR假结区域构建了一个嵌合TR（htTR）。当在体外与hTERT共表达时，嵌合RNA重建了人类端粒酶活性，但与hTR相比，在重复添加持续性和DNA合成水平上表现出缺陷。活性依赖于嵌合RNA中的tTR序列。htTR在体外与hTERT相互作用，并且主要通过其hTR主干的一个区域J7b/8a环二聚化。将htTR引入稳定表达hTERT的端粒酶阴性细胞中并不能重建能够延长端粒的活性酶。因此，我们的结果表明，嵌合RNA在体外重建了一种活性较弱的非持续性人类端粒酶，该酶在体内的端粒延长方面存在缺陷。这表明假结功能可能存在物种特异性要求。
