Genome-wide probing RNA structure with the modified DMS-MaPseq in Arabidopsis
Genome-wide probing RNA structure with the modified DMS-MaPseq in Arabidopsis
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DOI：
10.1016/j.ymeth.2018.11.018
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发表时间：
2019-02-15
期刊：
影响因子：
4.8
通讯作者：
Zhang, Xiuren
中科院分区：
文献类型：
--
作者：
Wang, Zhiye;Wang, Meiyue;Zhang, Xiuren
转录本具有形成稳定二级结构的内在倾向，这对于调节 RNA 转录、剪接、翻译、RNA 定位和周转至关重要。许多将化学反应与新一代测序 (NGS) 相结合的方法已被应用于研究体内 RNA 结构，为 RNA 生物学提供了新的见解。硫酸二甲酯 (DMS) 探测与 NGS 突变分析 (DMS-MaPseq) 相结合是一种新开发的方法，用于揭示全基因组或靶标特异性 RNA 结构。在此，我们提出了针对植物材料的改良 DMS-MaPseq 方法的实验方案。优化了DMS处理条件，简化了文库制备程序。我们还提供了用于全基因组 DMS-MaPseq 数据生物信息学分析的自定义脚本。生物信息学结果表明我们的方法可以生成高质量且可重复的数据。此外，我们评估了拟南芥全基因组 RNA 结构分析的测序深度和覆盖范围，并提供了移动 RNA 体内结构的两个例子。我们希望我们改进的 DMS-MaPseq 方法将成为分析植物体内 RNA 结构组的强大工具。
Transcripts have intrinsic propensity to form stable secondary structure that is fundamental to regulate RNA transcription, splicing, translation, RNA localization and turnover. Numerous methods that integrate chemical reactions with next-generation sequencing (NGS) have been applied to study in vivo RNA structure, providing new insights into RNA biology. Dimethyl sulfate (DMS) probing coupled with mutational profiling through NGS (DMS-MaPseq) is a newly developed method for revealing genome-wide or target-specific RNA structure. Herein, we present our experimental protocol of a modified DMS-MaPseq method for plant materials. The DMS treatment condition was optimized, and library preparation procedures were simplified. We also provided custom scripts for bioinformatic analysis of genome-wide DMS-MaPseq data. Bioinformatic results showed that our method could generate high-quality and reproducible data. Further, we assessed sequencing depth and coverage for genome-wide RNA structure profiling in Arabidopsis, and provided two examples of in vivo structure of mobile RNAs. We hope that our modified DMS-MaPseq method will serve as a powerful tool for analyzing in vivo RNA structurome in plants.