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Nature:华裔学者解码RNA调控机制

标签:RNA调控
摘要 : Chang教授及其同事首次开发了可以在活细胞中全面分析RNA二级结构的新技术,SHAPE(selective 2'-hydroxyl acylation and profiling experiment)。这一成果发表在近期的Nature杂志上。

 RNA在生物学系统中有着举足轻重的作用,它不仅将DNA的遗传信息传递给蛋白,还调控了各种生物学过程。单链RNA就像一条胶带,可以折叠起来通过碱基互补配对形成二级结构,这样的3D结构决定着RNA的功能。

用于结构分析的传统技术(比如X射线晶体衍射)一般比较麻烦,“而且一次只能用于一个RNA,”斯坦福大学的Howard Chang教授说。

为了同时研究多个RNA的二级结构,人们想到用化学修饰来区分未配对的核苷酸。通过RNA测序分析RNA的碱基修饰模式(即碱基配对状态),就可以知道RNA分子是如何折叠的。不过,这类全转录组分析技术还存在较大的局限:要么只能靶标四种核苷酸中的两个,要么只能用于体外研究。

Chang教授及其同事首次开发了可以在活细胞中全面分析RNA二级结构的新技术,SHAPE(selecive 2'-hydroxyl acylation and profiling experiment)。这一成果发表在近期的Nature杂志上。

Howard Chang教授曾入选HHMI2009年早期职业科学家项目(2009 Early Career Scientist Program),获得百万美元资助。这项计划会在6年中为每一位入选科学家提供包括薪酬,福利和研究预算在内的,超过150万美元的经费支持,这些科学家也会被聘为霍华德休斯医学研究院的全职研究员。近几年Howard Chang教授多次在顶级期刊上发表重要成果。

SHAPE分析首先是用NAI-N3修饰未配对的核苷酸,然后让生物素与NAI-N3结合,通过链霉亲和素磁珠回收修饰过的RNA。将这些RNA切割成小片段进行测序,可以揭示哪些核苷酸被修饰(也就是未配对),哪些核苷酸未被修饰(也就是配对或者结合蛋白)。

据介绍, SHAPE技术不仅可以在活细胞内修饰所有核苷酸,还可以富集被修饰的RNA,进一步提高信噪比。“现在我们可以在天然的细胞环境中,实现高分辨率、高通量的RNA分析,”康奈尔大学的Julius Lucks说。

研究人员还利用这一技术比较了RNA在细胞内和体外环境下的结构差异,鉴定了可能是蛋白结合位点的核苷酸序列。

原文标题:Structural imprints in vivo decode RNA regulatory mechanisms

原文摘要:Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program1. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA2, 3. Here we present a novel biochemical approach, in vivo click selective 2′-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA–protein interactions andN6-methyladenosine (m6A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

来源: Nature 浏览次数:0

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