通过DMS-MaPseqq检测到的活人细胞中的端粒酶RNA结构异质性
Nicholas M Forino1, Jia Zheng Woo2, Arthur J Zaug3,4
1Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, CA, USA.
bioRxiv : the preprint server for biology
|October 24, 2023
概括
人类端粒酶RNA (hTR) 错误折叠发生在细胞中,形成一个不活跃的结构. 这种替代的CR4/5形状阻碍了端粒酶的组合和活性,这表明其他辅助因子在端粒酶生物发生过程中发挥了作用.
科学领域:
- 分子生物学分子生物学
- 生物化学 生化学
- 遗传学 是一个遗传学.
背景情况:
- 端粒酶是维持端粒长度所必需的一种核糖蛋白酶.
- 人类端粒酶RNA (hTR) 是一个关键组成部分,为端粒DNA合成提供了模板.
- 包括CR4/5区域在内的hTR域的适当折叠对于结合端粒酶逆转录酶 (hTERT) 和催化活性至关重要.
研究的目的:
- 为了研究hTR的体内结构,特别是CR4/5域,使用二甲基硫酸盐突变分析与测序 (DMS-MaPseq).
- 确定存在于替代构造中的hTR的比例及其对端粒酶组合和功能的影响.
- 探索hTERT和其他潜在的辅助因子在hTR折叠和端粒酶生物发生中的作用.
主要方法:
- 用测序 (DMS-MaPseq) 进行二甲基硫酸盐突变分析,以探测活细胞中的RNA结构.
- 组合解卷分析来解释结构数据.
- 位点定向突变发生,以评估替代RNA构造的功能影响.
- 净化端粒酶核糖核蛋白 (RNP) 复合体.
主要成果:
- 大约15%的细胞hTR存在于另一种CR4/5形状,缺乏hTERT结合的特征.
- 这种替代形状是独立于hTERT表达的,并且不会被hTERT过度表达所纠正.
- 通过突变发生变异稳定替代CR4/5形状会损害端粒酶的组合和活性.
- 在纯化端粒酶RNP复合体中,替代的CR4/5形状不存在,这表明它不是活性形式.
结论:
- 细胞hTR的很大一部分可能存在于错误折叠,不活跃的状态,构成对端粒酶组装的动力障碍.
- 这些发现表明,hTERT以外的其他因素可能在确保telomerase生物发生过程中hTR正确折叠方面发挥作用.
- 这种错误折叠的hTR池很可能是缓慢重新折叠的,或者是降解的目标,这突显了精确的RNA in vivo折叠的重要性.
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