Maida Yoshiko, Yasukawa Mami, Ghilotti Marco, Ando Yoshinari, Masutomi Kenkichi
Division of Cancer Stem Cell, National Cancer Center Research Institute.
Division of Cancer Stem Cell, National Cancer Center Research Institute;
J Vis Exp. 2018 Jun 12(136):57021. doi: 10.3791/57021.
Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.
人端粒酶逆转录酶（TERT）是端粒酶的催化亚基，它通过依赖RNA的DNA聚合酶活性延长端粒。尽管TERT被命名为逆转录酶，但对TERT的结构和系统发育分析表明，TERT是右手聚合酶家族的成员，与病毒依赖RNA的RNA聚合酶（RdRP）以及病毒逆转录酶有关。我们首次鉴定了人TERT的RdRP活性，该活性可生成与模板非编码RNA互补的RNA链，并在癌细胞中促进RNA沉默。为了分析这种非经典酶活性，我们在2009年开发了用重组TERT进行的RdRP检测方法，此后又建立了针对内源性TERT的体外RdRP检测方法。在本手稿中，我们描述了后一种方法。简而言之，从细胞中分离出TERT免疫复合物，并与模板RNA和包括放射性rNTP在内的rNTP一起孵育以进行RdRP反应。为了去除单链RNA，用核糖核酸酶I处理反应产物，并用聚丙烯酰胺凝胶电泳分析最终产物。过夜曝光后，通过放射自显影可以检测到放射性标记的RdRP产物。
