Department of Biology, Johns Hopkins University, Baltimore, Maryland, USA
Department of Biology and Chemistry, Morehead State University, Morehead, Kentucky, USA.
Mol Cell Biol. 2020 Nov 20;40(24). doi: 10.1128/MCB.00239-20.
The telomerase ribonucleoprotein (RNP) counters the chromosome end replication problem, completing genome replication to prevent cellular senescence in yeast, humans, and most other eukaryotes. The telomerase RNP core enzyme is composed of a dedicated RNA subunit and a reverse transcriptase (telomerase reverse transcriptase [TERT]). Although the majority of the 1,157-nucleotide (nt) telomerase RNA, TLC1, is rapidly evolving, the central catalytic core is largely conserved, containing the template, template-boundary helix, pseudoknot, and core-enclosing helix (CEH). Here, we show that 4 bp of core-enclosing helix is required for telomerase to be active and to maintain yeast telomeres , whereas the ΔCEH and 1- and 2-bp alleles do not support telomerase function. Using the CRISPR/nuclease-deactivated Cas9 (dCas9)-based CARRY (CRISPR-assisted RNA-RNA-binding protein [RBP] yeast) two-hybrid assay to assess binding of our CEH mutant RNAs to TERT, we find that the 4-bp CEH RNA binds to TERT but the shorter-CEH constructs do not, consistent with the telomerase activity and complementation results. Thus, the CEH is essential in yeast telomerase RNA because it is needed to bind TERT to form the core RNP enzyme. Although the 8 nt that form this 4-bp stem at the base of the CEH are nearly invariant among species, our results with sequence-randomized and truncated-CEH helices suggest that this binding interaction with TERT is dictated more by secondary than by primary structure. In summary, we have mapped an essential binding site in telomerase RNA for TERT that is crucial to form the catalytic core of this biomedically important RNP enzyme.
端粒酶核糖核蛋白（RNP）解决了染色体末端复制问题，完成基因组复制，防止酵母、人类和大多数其他真核生物的细胞衰老。端粒酶 RNP 核心酶由专用 RNA 亚基和逆转录酶（端粒酶逆转录酶 [TERT]）组成。虽然 1157 个核苷酸（nt）的端粒酶 RNA TLC1 的大部分都在快速进化，但核心催化区大部分都保持保守，包含模板、模板结合螺旋、假结和核心封闭螺旋（CEH）。在这里，我们表明核心封闭螺旋的 4 个碱基对于端粒酶的活性和维持酵母端粒是必需的，而ΔCEH 和 1 个和 2 个碱基等位基因不支持端粒酶的功能。使用基于 CRISPR/核酸酶失活 Cas9（dCas9）的 CARRY（CRISPR 辅助 RNA-RNA 结合蛋白 [RBP] 酵母）双杂交测定法来评估我们的 CEH 突变 RNA 与 TERT 的结合，我们发现 4 个碱基的 CEH RNA 与 TERT 结合，但较短的 CEH 构建体不结合，与端粒酶活性和互补结果一致。因此，CEH 在酵母端粒酶 RNA 中是必不可少的，因为它需要与 TERT 结合形成核心 RNP 酶。尽管形成 CEH 底部的 4 个碱基的 8 个核苷酸在种间几乎不变，但我们对随机化和截断 CEH 螺旋的结果表明，这种与 TERT 的结合相互作用更多地取决于二级结构而不是一级结构。总之，我们已经在端粒酶 RNA 中映射了一个与 TERT 结合的必需结合位点，这对于形成这种具有重要生物医学意义的 RNP 酶的催化核心至关重要。
