Specificity and Stoichiometry of Subunit Interactions in the Human Telomerase Holoenzyme Assembled In Vivo
Specificity and Stoichiometry of Subunit Interactions in the Human Telomerase Holoenzyme Assembled In Vivo
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DOI：
10.1128/mcb.00151-10
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发表时间：
2010-06-01
影响因子：
5.3
通讯作者：
Collins, Kathleen
中科院分区：
文献类型：
--
作者：
Egan, Emily D.;Collins, Kathleen
人类端粒酶RNA （hTR）的H/ACA基序指导内源性端粒酶全酶组装、功能和调控的特定途径。hTR与其他H/ACA rna之间的相似性已经建立，但尽管hTR H/ACA RNP组装的独特特征导致人类疾病中端粒酶缺乏，但尚未探索差异。在这里，我们通过RNA积累、RNP亲和纯化和引物延伸活性测定来定义hTR H/ACA RNA和RNP结构。首先，我们评估了hTR H/ACA基序5'发夹的几种折叠模型。其次，我们证明了H/ACA RNA积累的5‘和3’发夹要求的意外和令人惊讶的普遍不对称性。第三，我们确定hTR组装了所有四种H/ACA RNP核心蛋白（dyskerin, NOP10， NHP2和GAR1）的两组而不是一组。第四，我们解决了hTR与全酶亚基WDR79/TCAB1相关的预测特异性差异。总之，这些结果完成了活性RNP生物发生所需的hTR元件的分析，并定义了所有功能必需的人类端粒酶全酶亚基的相互作用特异性和化学计量学。这项研究揭示了端粒酶和其他H/ACA RNPs之间意想不到的相似之处，但也存在差异，这使得端粒酶的生物发生和调节具有独特的特异性。
The H/ACA motif of human telomerase RNA (hTR) directs specific pathways of endogenous telomerase holoenzyme assembly, function, and regulation. Similarities between hTR and other H/ACA RNAs have been established, but differences have not been explored even though unique features of hTR H/ACA RNP assembly give rise to telomerase deficiency in human disease. Here, we define hTR H/ACA RNA and RNP architecture using RNA accumulation, RNP affinity purification, and primer extension activity assays. First, we evaluate alternative folding models for the hTR H/ACA motif 5' hairpin. Second, we demonstrate an unanticipated and surprisingly general asymmetry of 5' and 3' hairpin requirements for H/ACA RNA accumulation. Third, we establish that hTR assembles not one but two sets of all four of the H/ACA RNP core proteins, dyskerin, NOP10, NHP2, and GAR1. Fourth, we address a difference in predicted specificities of hTR association with the holoenzyme subunit WDR79/TCAB1. Together, these results complete the analysis of hTR elements required for active RNP biogenesis and define the interaction specificities and stoichiometries of all functionally essential human telomerase holoenzyme subunits. This study uncovers unexpected similarities but also differences between telomerase and other H/ACA RNPs that allow a unique specificity of telomerase biogenesis and regulation.