Gallardo Franck, Olivier Catherine, Dandjinou Alain T, Wellinger Raymund J, Chartrand Pascal
Département de Biochimie, Université de Montréal, Montréal, Quebec, Canada.
EMBO J. 2008 Mar 5;27(5):748-57. doi: 10.1038/emboj.2008.21. Epub 2008 Feb 14.
The yeast telomerase holoenzyme, which adds telomeric repeats at the chromosome ends, is composed of the TLC1 RNA and the associated proteins Est1, Est2 and Est3. To study the biogenesis of telomerase in endogenous conditions, we performed fluorescent in situ hybridization on the native TLC1 RNA. We found that the telomerase RNA colocalizes with telomeres in G1- to S-phase cells. Strains lacking any one of the Est proteins accumulate TLC1 RNA in their cytoplasm, indicating that a critical stage of telomerase biogenesis could take place outside of the nucleus. We were able to demonstrate that endogenous TLC1 RNA shuttles between the nucleus and the cytoplasm, in association with the Crm1p exportin and the nuclear importins Mtr10p-Kap122p. Furthermore, nuclear retention of the TLC1 RNA is impaired in the absence of yKu70p, Tel1p or the MRX complex, which recruit telomerase to telomeres. Altogether, our results reveal that the nucleo-cytoplasmic trafficking of the TLC1 RNA is an important step in telomere homeostasis, and link telomerase biogenesis to its recruitment to telomeres.
酵母端粒酶全酶在染色体末端添加端粒重复序列,由TLC1 RNA以及相关蛋白Est1、Est2和Est3组成。为了在内源条件下研究端粒酶的生物发生,我们对天然TLC1 RNA进行了荧光原位杂交。我们发现端粒酶RNA在G1期到S期细胞中与端粒共定位。缺乏任何一种Est蛋白的菌株会在其细胞质中积累TLC1 RNA,这表明端粒酶生物发生的一个关键阶段可能发生在细胞核外。我们能够证明内源性TLC1 RNA与Crm1p输出蛋白和核输入蛋白Mtr10p-Kap122p结合,在细胞核和细胞质之间穿梭。此外,在缺乏将端粒酶招募到端粒的yKu70p、Tel1p或MRX复合物的情况下,TLC1 RNA的核滞留受损。总之,我们的结果表明TLC1 RNA的核质运输是端粒稳态的一个重要步骤,并将端粒酶的生物发生与其招募到端粒联系起来。