Lead-seq: transcriptome-wide structure probing in vivo using lead(II) ions

被引:20
|
作者
Twittenhoff, Christian [1 ]
Brandenburg, Vivian B. [1 ]
Righetti, Francesco [1 ]
Nuss, Aaron M. [2 ]
Mosig, Axel [3 ]
Dersch, Petra [2 ,4 ]
Narberhaus, Franz [1 ]
机构
[1] Ruhr Univ Bochum, Microbial Biol, D-44780 Bochum, Germany
[2] Helmholtz Ctr Infect Res, Dept Mol Infect Biol, D-381214 Braunschweig, Germany
[3] Ruhr Univ Bochum, Dept Biophys, D-44780 Bochum, Germany
[4] Univ Munster, Inst Infectiol, Ctr Mol Biol Inflammat, D-48149 Munster, Germany
关键词
RNA SECONDARY STRUCTURE; SELECTIVE 2'-HYDROXYL ACYLATION; RIBOSOMAL-RNA; MOLECULAR CHAPERONES; CHEMICAL PROBES; P-RNA; SHAPE; CLEAVAGE; REVEALS; BINDING;
D O I
10.1093/nar/gkaa404
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 pseudo-tuberculosis, 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. pseu-dotuberculosis 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.
引用
收藏
页码:E71 / E71
页数:19
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