Twittenhoff Christian, Brandenburg Vivian B, Righetti Francesco, Nuss Aaron M, Mosig Axel, Dersch Petra, Narberhaus Franz
Microbial Biology, Ruhr University Bochum, 44780 Bochum, Germany.
Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, 381214 Braunschweig, Germany.
Nucleic Acids Res. 2020 Jul 9;48(12):e71. doi: 10.1093/nar/gkaa404.
The dynamic conformation of RNA molecules within living cells is key to their function. Recent advances in probing the RNA structurome in vivo, including the use of SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) or kethoxal reagents or DMS (dimethyl sulfate), provided unprecedented insights into the architecture of RNA molecules in the living cell. Here, we report the establishment of lead probing in a global RNA structuromics approach. In order to elucidate the transcriptome-wide RNA landscape in the enteric pathogen Yersinia pseudotuberculosis, we combined lead(II) acetate-mediated cleavage of single-stranded RNA regions with high-throughput sequencing. This new approach, termed 'Lead-seq', provides structural information independent of base identity. We show that the method recapitulates secondary structures of tRNAs, RNase P RNA, tmRNA, 16S rRNA and the rpsT 5'-untranslated region, and that it reveals global structural features of mRNAs. The application of Lead-seq to Y. pseudotuberculosis cells grown at two different temperatures unveiled the first temperature-responsive in vivo RNA structurome of a bacterial pathogen. The translation of candidate genes derived from this approach was confirmed to be temperature regulated. Overall, this study establishes Lead-seq as complementary approach to interrogate intracellular RNA structures on a global scale.
活细胞内RNA分子的动态构象是其发挥功能的关键。近年来，在体内探测RNA结构组方面取得了进展，包括使用SHAPE（通过引物延伸分析的选择性2'-羟基酰化）、乙二醛试剂或DMS（硫酸二甲酯），这为活细胞中RNA分子的结构提供了前所未有的见解。在此，我们报告了在全球RNA结构组学方法中建立铅离子探测技术。为了阐明肠道病原体假结核耶尔森菌全转录组范围的RNA概况，我们将乙酸铅介导的单链RNA区域切割与高通量测序相结合。这种新方法称为“Lead-seq”，可提供独立于碱基身份的结构信息。我们表明，该方法概括了tRNA、核糖核酸酶P RNA、转移信使RNA、16S rRNA和rpsT 5'-非翻译区的二级结构，并揭示了mRNA的整体结构特征。将Lead-seq应用于在两种不同温度下生长的假结核耶尔森菌细胞，揭示了细菌病原体首个温度响应性的体内RNA结构组。通过这种方法获得的候选基因的翻译被证实受温度调节。总体而言，本研究确立了Lead-seq作为在全球范围内研究细胞内RNA结构的补充方法。
