Comparison of 5' UTR structures in Listeria monocytogenes

To understand physiological processes in vivo, it is vital to disclose the conformation of biological molecules inside living cells and correlate their structure with their function. Here, we describe a method, FUSE (5’-UTR Structure Elucidation) that was used to characterize changes in the structures of 5’-UTRs at different conditions inside the human bacterial pathogen Listeria monocytogenes. Using FUSE, we discovered a novel RNA thermoswitch and calculated the ratio of two conformations that this thermoswitch assumes at different conditions. FUSE could also identify the site of base-pairing interaction between a small RNA and mRNA with a single-nucleotide resolution. Strikingly, FUSE discovered a functional mRNA-mRNA interaction, where the stability of mRNA encoding PrsA2 chaperone, was increased by the direct binding of an mRNA encoding its substrate, listeriolysin O, the main L. monocytogenes virulence factor. Owing to its focus on 5’-UTRs, FUSE can be applied to analyse these regulatory regions in any living organism. L. monocytogenes EGD-e cells or RNA isolated from these cells were treated with dimethyl sulphate (DMS). The modified RNA served as a template for cDNA library construction according to FUSE (5' UTR Structure Elucidation) or Structure-seq protocols. Both these methods employ TGIRTIII reverse transcriptase to encode methylated adenines and cytosines as mismatches in cDNA sequence. While Structure-seq protocol synthesizes cDNA from the total RNA, FUSE selectively enriches for the 5' UTR regions in the cDNA library. The resulting cDNA libraries were sequenced on Illumina MiSeq platform in 2 x 75 bp paired-end mode.为了在活体水平解析生理过程，揭示活细胞内生物分子的构象并将其结构与功能建立关联，这一点至关重要。本文描述了一种名为FUSE（5’-UTR结构解析法，5’-UTR Structure Elucidation）的方法，该方法可用于表征人类细菌性致病菌单核细胞增生李斯特菌（Listeria monocytogenes）内不同条件下5’-非翻译区（5’-UTR）的结构变化。借助FUSE技术，我们发现了一种新型RNA热开关，并计算出该热开关在不同条件下两种构象的占比。FUSE还能够以单核苷酸分辨率鉴定小RNA（small RNA）与信使RNA（mRNA）之间的碱基配对相互作用位点。尤为值得关注的是，FUSE揭示了一种功能性的mRNA-mRNA相互作用：编码PrsA2分子伴侣的mRNA，其稳定性可通过编码其底物——李斯特溶素O（listeriolysin O，单核细胞增生李斯特菌的主要毒力因子）的mRNA的直接结合而得到显著提升。由于该技术聚焦于5’-UTR区域，FUSE可应用于分析任意活生物体中的此类基因调控区域。我们对单核细胞增生李斯特菌EGD-e菌株的细胞，或从这些细胞中分离得到的RNA，使用硫酸二甲酯（DMS）进行化学修饰。随后，根据FUSE（5’-UTR结构解析法）或Structure-seq实验方案，以修饰后的RNA为模板构建互补DNA（cDNA）文库。这两种方法均采用TGIRTIII逆转录酶，将甲基化修饰的腺嘌呤与胞嘧啶编码为cDNA序列中的错配碱基。尽管Structure-seq方案以总RNA为模板合成cDNA，但FUSE可在最终的cDNA文库中选择性富集5’-UTR区域。最终得到的cDNA文库在Illumina MiSeq测序平台上以2×75 bp双端测序模式完成测序。