A 4-Base-Pair Core-Enclosing Helix in Telomerase RNA Is Essential for Activity and for Binding to the Telomerase Reverse Transcriptase Catalytic Protein Subunit.
A 4-Base-Pair Core-Enclosing Helix in Telomerase RNA Is Essential for Activity and for Binding to the Telomerase Reverse Transcriptase Catalytic Protein Subunit.
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DOI：
10.1128/mcb.00239-20
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发表时间：
2020-11-20
影响因子：
5.3
通讯作者：
Zappulla DC
中科院分区：
文献类型：
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作者：
Mefford MA;Hass EP;Zappulla DC
端粒酶核糖核蛋白（RNP）对抗染色体末端复制问题，完成基因组复制，以防止酵母，人类和大多数其他真核生物的细胞衰老。端粒酶RNP核心酶由一个专门的RNA亚基和一个逆转录酶（端粒酶逆转录酶[TERT]）组成。虽然大多数1，157个核苷酸（nt）的酿酒酵母端粒酶RNA（TLC 1）正在迅速进化，但中心催化核心在很大程度上是保守的，包含模板，模板边界螺旋，假结和核心包围螺旋（CEH）。端粒酶核糖核蛋白（RNP）对抗染色体末端复制问题，完成基因组复制，以防止酵母，人类和大多数其他真核生物的细胞衰老。端粒酶RNP核心酶由一个专门的RNA亚基和一个逆转录酶（端粒酶逆转录酶[TERT]）组成。虽然大多数1，157个核苷酸（nt）的酿酒酵母端粒酶RNA（TLC 1）正在迅速进化，但中心催化核心在很大程度上是保守的，包含模板，模板边界螺旋，假结和核心包围螺旋（CEH）。在这里，我们发现，4 bp的核心包围螺旋是端粒酶在体外活性和维持酵母端粒在体内所必需的，而ΔCEH和1-和2-bp的等位基因不支持端粒酶功能。使用基于CRISPR/核酸酶失活的Cas9（dCas 9）的CARRY（CRISPR辅助的RNA-RNA结合蛋白[RBP]酵母）双杂交测定来评估我们的CEH突变体RNA与TERT的结合，我们发现4-bp CEH RNA与TERT结合，但较短的CEH构建体不与TERT结合，这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中是必不可少的，因为它需要结合TERT以形成核心RNP酶。虽然在CEH的基础上形成这个4-bp茎的8个核苷酸在酵母属物种中几乎是不变的，但我们的序列随机化和截短CEH螺旋的结果表明，这种与TERT的结合相互作用更多地由二级结构决定，而不是由一级结构决定。总之，我们已经在端粒酶RNA中绘制了一个用于TERT的必需结合位点，该位点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。
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). 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.