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) Saccharomyces cerevisiae 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 in vitro and to maintain yeast telomeres in vivo, 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 in vivo 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 Saccharomyces 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.

中文翻译：

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端粒酶RNA中的4-碱基对核心封闭螺旋对于活性和与端粒酶逆转录酶催化蛋白亚基的结合至关重要

端粒酶核糖核蛋白（RNP）解决了染色体末端复制问题，完成了基因组复制以防止酵母，人类和大多数其他真核生物中的细胞衰老。端粒酶RNP核心酶由专用的RNA亚基和逆转录酶（端粒酶逆转录酶[TERT]）组成。尽管大多数1,157个核苷酸（nt）酿酒酵母端粒酶RNA TLC1迅速发展，但中央催化核心在很大程度上是保守的，包含模板，模板边界螺旋，假结和核心封闭螺旋（CEH）。在这里，我们显示端粒酶在体外具有活性并在体内维持酵母端粒需要4 bp的核心封闭螺旋，而ΔCEH和1和2 bp等位基因不支持端粒酶功能。使用基于CRISPR /核酸酶的Cas9（dCas9）灭活的CARRY（CRISPR辅助RNA–RNA结合蛋白[RBP]酵母）两种杂交测定法评估我们的CEH突变RNA与TERT的结合，我们发现4- bp CEH RNA与TERT结合，但较短的CEH构建体不结合，与端粒酶活性和体内互补结果一致。因此，CEH在酵母端粒酶RNA中必不可少，因为它需要与TERT结合形成核心RNP酶。尽管在酵母菌的底端形成4 bp茎的8 nt在酵母菌中几乎不变物种，我们与序列随机和截断的CEH螺旋的结果表明，与TERT的这种结合相互作用更多地是由二级结构决定，而不是由一级结构决定。总之，我们在端粒酶RNA中为TE​​RT绘制了一个必不可少的结合位点，这对于形成这种生物医学上重要的RNP酶的催化核心至关重要。