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Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo

机译：全基因组的RNA结构探测揭示了体内mRNA结构的主动展开

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摘要

RNA has a dual role as an informational molecule and a direct effector of biological tasks. The latter function is enabled by RNA's ability to adopt complex secondary and tertiary folds and thus has motivated extensive computational and experimental efforts for determining RNA structures. Existing approaches for evaluating RNA structure have been largely limited to in vitro systems, yet the thermodynamic forces which drive RNA folding in vitro may not be sufficient to predict stable RNA structures in vivo. Indeed, the presence of RNA-binding proteins and ATP-dependent heli-cases can influence which structures are present inside cells. Here we present an approach for globally monitoring RNA structure in native conditions in vivo with single-nucleotide precision. This method is based on in vivo modification with dimethyl sulphate (DMS), which reacts with unpaired adenine and cytosine residues, followed by deep sequencing to monitor modifications. Our data from yeast and mammalian cells are in excellent agreement with known messenger RNA structures and with the high-resolution crystal structure of the Saccharomyces cerevisiae ribosome. Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro. Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast shows that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells. Our studies broadly enable the functional analysis of physiological RNA structures and reveal that, in contrast to the Anfinsen view of protein folding whereby the structure formed is the most thermodynami-cally favourable, thermodynamics have an incomplete role in determining mRNA structure in vivo.
机译：RNA具有信息分子和生物学任务的直接效应器的双重作用。后一种功能是由于RNA能够采用复杂的二级和三级折叠而实现的，因此激发了广泛的计算和实验工作来确定RNA结构。现有的用于评估RNA结构的方法主要限于体外系统，但是驱动RNA在体外折叠的热力学力可能不足以预测体内稳定的RNA结构。实际上，RNA结合蛋白和ATP依赖的heli-case的存在会影响细胞内部存在的结构。在这里，我们提出了一种在自然条件下以单核苷酸精度整体监测RNA结构的方法。此方法基于硫酸二甲酯（DMS）的体内修饰，该修饰与未配对的腺嘌呤和胞嘧啶残基反应，然后进行深度测序以监测修饰。我们从酵母和哺乳动物细胞获得的数据与已知的信使RNA结构以及酿酒酵母核糖体的高分辨率晶体结构极为吻合。体内和体外数据之间的比较表明，在快速分裂的细胞中，体内的结构化mRNA区域远少于体外。甚至热稳定的RNA结构也经常在细胞中变性，突出了细胞过程在调节RNA结构中的重要性。确实，在酵母中ATP耗尽的条件下对mRNA结构的分析表明，依赖能量的过程强烈促进了细胞内mRNA的主要展开状态。我们的研究广泛地实现了生理学RNA结构的功能分析，并揭示了与蛋白折叠的Anfinsen观点相反，后者形成的结构是最热动力学有利的，而热力学在确定体内mRNA结构方面不完全起作用。

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  • 来源
    《Nature》 |2014年第7485期|701-705|共5页
  • 作者

    Silvi Rouskin; Meghan Zubradt; Stefan Washietl; Manolis Kellis; Jonathan S. Weissman;

  • 作者单位

    Department of Cellular and Molecular Pharmacology, California Institute of Quantitative Biology, Center for RNA Systems Biology, Howard Hughes Medical Institute, University of California, San Francisco, California 94158, USA;

    Department of Cellular and Molecular Pharmacology, California Institute of Quantitative Biology, Center for RNA Systems Biology, Howard Hughes Medical Institute, University of California, San Francisco, California 94158, USA;

    Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA,Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA,The Broad Institute, Cambridge, Massachusetts 02139, USA;

    Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA,Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA,The Broad Institute, Cambridge, Massachusetts 02139, USA;

    Department of Cellular and Molecular Pharmacology, California Institute of Quantitative Biology, Center for RNA Systems Biology, Howard Hughes Medical Institute, University of California, San Francisco, California 94158, USA;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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