Chemical modifications in RNAs play crucial roles in diversifying their structures and regulating numerous biochemical processes. Since the 1990s, several hydrophobic prenyl-modifications have been discovered in various RNAs. Prenyl groups serve as precursors for terpenes and many other biological molecules. The processes of prenylation in different macromolecules have been extensively studied. We introduce here a novel chemical biology toolkit that not only labels i6A, a prenyl-modified RNA residue, by leveraging the unique reactivity of the prenyl group, but also provides a general strategy to incorporate fluorescence functionalities into RNAs for molecular tracking purposes. Our findings revealed that iodine-mediated cyclization reactions of the prenyl group occur rapidly, transforming i6A from a hydrogen-bond acceptor to a donor. Based on this reactivity, we developed an Iodine-Mediated Cyclization and Reverse Transcription (IMCRT) tRNA-seq method, which can profile all nine endogenous tRNAs containing i6A residues in Saccharomyces cerevisiae with single-base resolution. Furthermore, under stress conditions, we observed a decline in i6A levels in budding yeast, accompanied by significant decrease of mutation rate at A37 position. Thus, the IMCRT tRNA-seq method not only permits semi-quantification of i6A levels in tRNAs but also holds potential for transcriptome-wide detection and analysis of various RNA species containing i6A modifications.

中文翻译：

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N6-异戊烯基腺苷 (i6A) 修饰 RNA 的生物正交标记和分析

RNA 的化学修饰在使其结构多样化和调节众多生化过程中发挥着至关重要的作用。自 20 世纪 90 年代以来，在各种 RNA 中发现了几种疏水性异戊二烯基修饰。异戊二烯基团是萜烯和许多其他生物分子的前体。不同大分子中的异戊二烯化过程已被广泛研究。我们在这里介绍一种新型化学生物学工具包，它不仅通过利用异戊二烯基团的独特反应性来标记 i6A（一种异戊二烯基修饰的 RNA 残基），而且还提供了将荧光功能整合到 RNA 中以进行分子追踪的通用策略。我们的研究结果表明，碘介导的异戊烯基环化反应迅速发生，将 i6A 从氢键受体转变为供体。基于这种反应性，我们开发了一种碘介导的环化和反转录（IMCRT）tRNA-seq方法，该方法可以以单碱基分辨率分析酿酒酵母中所有九种含有i6A残基的内源tRNA。此外，在胁迫条件下，我们观察到芽殖酵母中 i6A 水平下降，同时 A37 位点突变率显着下降。因此，IMCRT tRNA-seq 方法不仅可以半定量 tRNA 中的 i6A 水平，而且还具有对包含 i6A 修饰的各种 RNA 进行全转录组检测和分析的潜力。