RNA structure plays roles in myriad cellular events including transcription, translation, and RNA processing. Genome-wide analyses of RNA secondary structure in vivo by chemical probing have revealed critical structural features of mRNAs and long ncRNAs. Here, we examine the in vivo secondary structure of a small RNA class, tRNAs. Study of tRNA structure is challenging because tRNAs are heavily modified and strongly structured. We introduce "tRNA structure-seq," a new workflow that accurately determines in vivo secondary structures of tRNA. The workflow combines dimethyl sulfate (DMS) probing, ultra-processive RT, and mutational profiling (MaP), which provides mutations opposite DMS and natural modifications thereby allowing multiple modifications to be identified in a single read. We applied tRNA structure-seq to E. coli under control and stress conditions. A leading folding algorithm predicts E. coli tRNA structures with only ∼80% average accuracy from sequence alone. Strikingly, tRNA structure-seq, by providing experimental restraints, improves structure prediction under in vivo conditions to ∼95% accuracy, with more than 14 tRNAs predicted completely correctly. tRNA structure-seq also quantifies the relative levels of tRNAs and their natural modifications at single nucleotide resolution, as validated by LC-MS/MS. Our application of tRNA structure-seq yields insights into tRNA structure in living cells, revealing that it is not immutable but has dynamics, with partial unfolding of secondary and tertiary tRNA structure under heat stress that is correlated with a loss of tRNA abundance. This method is applicable to other small RNAs, including those with natural modifications and highly structured regions.

中文翻译：

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体内 tRNA 结构组的全基因组分析揭示了热应激下的 RNA 结构和修饰动力学。

RNA 结构在无数细胞事件中发挥作用，包括转录、翻译和 RNA 加工。通过化学探测对体内 RNA 二级结构进行的全基因组分析揭示了 mRNA 和长 ncRNA 的关键结构特征。在这里，我们检查了小 RNA 类 tRNA 的体内二级结构。对 tRNA 结构的研究具有挑战性，因为 tRNA 经过大量修改且结构坚固。我们介绍了“tRNA structure-seq”，这是一种新的工作流程，可以准确确定 tRNA 的体内二级结构。该工作流程结合了硫酸二甲酯 (DMS) 探测、超处理 RT 和突变分析 (MaP)，它提供了与 DMS 和自然修饰相反的突变，从而允许在一次读取中识别多个修饰。我们将 tRNA structure-seq 应用于大肠杆菌。大肠杆菌在控制和压力条件下。领先的折叠算法仅根据序列预测大肠杆菌 tRNA 结构的平均准确度仅为 ~80%。引人注目的是，tRNA structure-seq 通过提供实验限制，将体内条件下的结构预测提高到 95% 左右的准确度，超过 14 个 tRNA 的预测完全正确。tRNA structure-seq 还量化了 tRNA 的相对水平及其在单核苷酸分辨率下的自然修饰，如 LC-MS/MS 所验证的那样。我们对 tRNA structure-seq 的应用深入了解了活细胞中的 tRNA 结构，表明它并非一成不变，而是具有动态性，在热应激下二级和三级 tRNA 结构的部分展开与 tRNA 丰度的损失相关。此方法适用于其他小RNA，