Etheridge Katherine T, Banik Soma S R, Armbruster Blaine N, Zhu Yusheng, Terns Rebecca M, Terns Michael P, Counter Christopher M
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 2002 Jul 5;277(27):24764-70. doi: 10.1074/jbc.M201227200. Epub 2002 Apr 15.
Telomerase is the enzyme essential to complete the replication of the terminal DNA of most eukaryotic chromosomes. In humans, this enzyme is composed of the telomerase reverse transcriptase (hTERT) and telomerase RNA (hTR) subunits. hTR has been found in the nucleolus, a site of assembly of ribosomes as well as other ribonucleoproteins (RNPs). We therefore tested whether the hTERT component is also found in the nucleolus, where it could complex with the hTR RNA to form a functional enzyme. We report here that hTERT does indeed localize to the nucleolus, and we mapped the domain responsible for this localization to the hTR-binding region of the protein by deletion analysis. Substitution mutations in two of the three conserved hTR-binding domains in this nucleolar localization domain (NoLD) abolished nucleolar localization. However, another mutation that impeded hTR binding did not alter this subcellular localization. Additionally, wild type hTERT was detected in the nucleolus of cells that failed to express hTR. Taken together, we propose that the nucleolar localization of hTERT involves more than just the association with the hTR subunit. Furthermore, the coincidental targeting of both the hTR and hTERT subunits to the nucleolus supports the premise that the assembly of telomerase occurs in the nucleolus.
端粒酶是完成大多数真核染色体末端DNA复制所必需的酶。在人类中，这种酶由端粒酶逆转录酶（hTERT）和端粒酶RNA（hTR）亚基组成。hTR已在核仁中被发现，核仁是核糖体以及其他核糖核蛋白（RNP）的组装位点。因此，我们测试了hTERT组分是否也存在于核仁中，在那里它可能与hTR RNA结合形成一种功能性酶。我们在此报告hTERT确实定位于核仁，并且通过缺失分析将负责这种定位的结构域定位到该蛋白的hTR结合区域。在这个核仁定位结构域（NoLD）中三个保守的hTR结合结构域中的两个发生取代突变后，核仁定位被消除。然而，另一个阻碍hTR结合的突变并没有改变这种亚细胞定位。此外，在未能表达hTR的细胞的核仁中检测到野生型hTERT。综上所述，我们提出hTERT的核仁定位不仅仅涉及与hTR亚基的结合。此外，hTR和hTERT亚基同时靶向核仁支持了端粒酶组装发生在核仁中的前提。
