Department of Chemistry, Pennsylvania State University, University Park, PA 16802.
Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802.
Proc Natl Acad Sci U S A. 2022 Jun 21;119(25):e2201237119. doi: 10.1073/pnas.2201237119. Epub 2022 Jun 13.
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 under control and stress conditions. A leading folding algorithm predicts 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.
RNA 结构在包括转录、翻译和 RNA 处理在内的众多细胞事件中发挥作用。通过化学探测对体内 RNA 二级结构的全基因组分析揭示了 mRNAs 和长非编码 RNA 的关键结构特征。在这里，我们研究了一类小 RNA，tRNA 的体内二级结构。研究 tRNA 结构具有挑战性，因为 tRNA 受到强烈的修饰和结构约束。我们引入了“tRNA 结构-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、超连续 RT 和突变分析（MaP），这提供了与 DMS 相反的突变和天然修饰，从而可以在单个读取中识别多个修饰。我们在对照和应激条件下应用 tRNA 结构-seq。一种领先的折叠算法仅根据序列预测 tRNA 结构的准确率约为 80%。引人注目的是，tRNA 结构-seq 通过提供实验约束，将体内条件下的结构预测准确性提高到约 95%，其中超过 14 个 tRNA 被完全正确预测。tRNA 结构-seq 还以单核苷酸分辨率定量 tRNA 的相对水平及其天然修饰，这通过 LC-MS/MS 得到了验证。我们对 tRNA 结构-seq 的应用揭示了活细胞中 tRNA 结构的一些见解，表明它不是一成不变的，而是具有动态性，在热应激下二级和三级 tRNA 结构部分展开，与 tRNA 丰度的损失相关。这种方法适用于其他小 RNA，包括具有天然修饰和高度结构区域的小 RNA。
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