In-cell RNA structure probing with SHAPE-MaP.
In-cell RNA structure probing with SHAPE-MaP.
复制标题
DOI：
10.1038/nprot.2018.010
复制
发表时间：
2018-06
期刊：
影响因子：
14.8
通讯作者：
Weeks KM
中科院分区：
文献类型：
--
作者：
Smola MJ;Weeks KM
RNA在许多细胞过程中起着关键作用。RNA的基本结构是转录本如何发挥功能、如何加工以及如何与RNA结合蛋白和配体相互作用的重要决定因素。通过引物延伸（SHAPE）分析的选择性2 '-羟基酰化的RNA结构分析利用与2'-羟基反应的小亲电化学探针的反应性来评估核苷酸分辨率下的RNA结构。当与突变分析（MaP）结合时，其中修饰的核苷酸在逆转录过程中被检测为内部错误编码，然后通过大规模平行测序读出，SHAPE产生RNA结构的定量每个核苷酸测量。在这里，我们提供了我们以前的体外SHAPE-MaP协议的扩展，并提供了在活细胞中进行和分析SHAPE-MaP探测实验的详细指导。MaP策略适用于丰富的转录组实验和低至中等丰度的细胞RNA，这些RNA不能通过全转录组方法很好地检测。细胞内SHAPE-MaP在大约3天内完成，可应用于从细菌到培养的哺乳动物细胞的细胞类型，并与各种结构探测试剂兼容。我们详细介绍了几种策略，通过这些策略，细胞内SHAPE-MaP可以告知新的生物学假设，并强调下游分析，揭示细胞中RNA相互作用的重要序列或结构基序。在这个协议扩展中，作者详细介绍了他们以前的体外SHAPE-MaP协议的细胞内版本，使RNA结构能够在活细胞中探测。TWEET：SHAPE-MaP探测细胞内RNA结构
RNAs play key roles in many cellular processes. The underlying structure of RNA is an important determinant for how transcripts function, are processed, and interact with RNA-binding proteins and ligands. RNA structure analysis by selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) takes advantage of the reactivity of small electrophilic chemical probes that react with the 2’-hydroxyl group to assess RNA structure at nucleotide resolution. When coupled with mutational profiling (MaP), in which modified nucleotides are detected as internal miscodings during reverse transcription and then read out by massively parallel sequencing, SHAPE yields quantitative per-nucleotide measurements of RNA structure. Here, we provide an Extension to our previous in vitro SHAPE-MaP protocol with detailed guidance for undertaking and analyzing SHAPE-MaP probing experiments in live cells. The MaP strategy works for both abundant-transcriptome experiments and for cellular RNAs of low to moderate abundance, which are not well examined by whole-transcriptome methods. In-cell SHAPE-MaP, performed in roughly 3 days, can be applied in cell types ranging from bacteria to cultured mammalian cells and is compatible with a variety of structure-probing reagents. We detail several strategies by which in-cell SHAPE-MaP can inform new biological hypotheses and emphasize downstream analyses that reveal sequence or structure motifs important for RNA interactions in cells. In this Protocol Extension, the Authors detail an in-cell version of their previous in vitro SHAPE-MaP protocol, enabling RNA structure to be probed in living cells. TWEET: In-cell RNA structure probing with SHAPE-MaP
登录
查看更多内容
影响因子：
7.7
作者：
Kondo Y;Oubridge C;van Roon AM;Nagai K
通讯作者：
Nagai K
DOI：
10.1261/rna.058586.116
发表时间：
2017-01
期刊：
RNA (New York, N.Y.)
影响因子：
--
作者：
Krokhotin A;Mustoe AM;Weeks KM;Dokholyan NV
通讯作者：
Dokholyan NV
DOI：
10.3390/v3091739
发表时间：
2011-09
期刊：
Viruses
影响因子：
--
作者：
Gebhard LG;Filomatori CV;Gamarnik AV
通讯作者：
Gamarnik AV
影响因子：
5.4
作者：
Kuhlmann, Micki M.;Chattopadhyay, Maitreyi;Simon, Anne E.
通讯作者：
Simon, Anne E.
影响因子：
4.5
作者：
Fang R;Moss WN;Rutenberg-Schoenberg M;Simon MD
通讯作者：
Simon MD