Queuosine (Q) is a conserved tRNA modification at the wobble anticodon position of tRNAs that read the codons of amino acids Tyr, His, Asn, and Asp. Q-modification in tRNA plays important roles in the regulation of translation efficiency and fidelity. Queuosine tRNA modification is synthesized de novo in bacteria, whereas in mammals the substrate for Q-modification in tRNA is queuine, the catabolic product of the Q-base of gut bacteria. This gut microbiome dependent tRNA modification may play pivotal roles in translational regulation in different cellular contexts, but extensive studies of Q-modification biology are hindered by the lack of high throughput sequencing methods for its detection and quantitation. Here, we describe a periodate-treatment method that enables single base resolution profiling of Q-modification in tRNAs by Nextgen sequencing from biological RNA samples. Periodate oxidizes the Q-base, which results in specific deletion signatures in the RNA-seq data. Unexpectedly, we found that periodate-treatment also enables the detection of several 2-thio-modifications including τm5s2U, mcm5s2U, cmnm5s2U, and s2C by sequencing in human and E. coli tRNA. We term this method periodate-dependent analysis of queuosine and sulfur modification sequencing (PAQS-seq). We assess Q- and 2-thio-modifications at the tRNA isodecoder level, and 2-thio modification changes in stress response. PAQS-seq should be widely applicable in the biological studies of Q- and 2-thio-modifications in mammalian and microbial tRNAs.

中文翻译：

---

通过高通量测序分析 queuosine 和 2-thio tRNA 修饰

Queuosine (Q) 是 tRNA 摆动反密码子位置上的保守 tRNA 修饰，可读取氨基酸 Tyr、His、Asn 和 Asp 的密码子。tRNA 中的 Q 修饰在翻译效率和保真度的调节中发挥着重要作用。queuosine tRNA 修饰是在细菌中从头合成的，而在哺乳动物中，tRNA 中 Q 修饰的底物是 queuine，它是肠道细菌 Q 碱基的分解代谢产物。这种肠道微生物组依赖性 tRNA 修饰可能在不同细胞背景下的翻译调控中发挥关键作用，但由于缺乏用于检测和定量的高通量测序方法，对 Q 修饰生物学的广泛研究受到阻碍。在这里，我们描述了一种高碘酸盐处理方法，该方法能够通过生物 RNA 样本的 Nextgen 测序对 tRNA 中的 Q 修饰进行单碱基分辨率分析。高碘酸盐氧化 Q 碱基，从而在 RNA-seq 数据中产生特定的删除特征。出乎意料的是，我们发现高碘酸盐处理还可以通过对人和大肠杆菌 tRNA 进行测序来检测多种 2-硫代修饰，包括 τm5s2U、mcm5s2U、cmnm5s2U 和 s2C。我们将这种方法称为高碘酸依赖的队列和硫修饰测序分析 (PAQS-seq)。我们评估了 tRNA 同解码器水平上的 Q- 和 2-硫代修饰，以及应激反应中的 2-硫代修饰变化。PAQS-seq 应广泛应用于哺乳动物和微生物 tRNA 中 Q- 和 2-硫代修饰的生物学研究。