Department of Chemistry, Princeton University, Princeton, NJ, 08544, USA.
Nat Commun. 2022 Jul 19;13(1):4176. doi: 10.1038/s41467-022-31876-2.
Epitranscriptomic RNA modifications can regulate fundamental biological processes, but we lack approaches to map modification sites and probe writer enzymes. Here we present a chemoproteomic strategy to characterize RNA 5-methylcytidine (mC) dioxygenase enzymes in their native context based upon metabolic labeling and activity-based crosslinking with 5-ethynylcytidine (5-EC). We profile mC dioxygenases in human cells including ALKBH1 and TET2 and show that ALKBH1 is the major hmC- and fC-forming enzyme in RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-iCLIP and ARP-based sequencing to identify ALKBH1-dependent mC oxidation in a variety of tRNAs and mRNAs and analyze ALKBH1 substrate specificity in vitro. We also apply targeted pyridine borane-mediated sequencing to measure fC sites on select tRNA. Finally, we show that fC at the wobble position of tRNA-Leu-CAA plays a role in decoding Leu codons under stress. Our work provides powerful chemical approaches for studying RNA mC dioxygenases and mapping oxidative mC modifications and reveals the existence of novel epitranscriptomic pathways for regulating RNA function.
表观转录组 RNA 修饰可以调节基本的生物学过程，但我们缺乏绘制修饰位点和探测写入酶的方法。在这里，我们提出了一种基于代谢标记和 5-乙炔胞嘧啶（5-EC）活性的化学生物组学策略，用于在天然环境中对 RNA 5-甲基胞嘧啶（mC）双加氧酶进行表征。我们在人细胞中对 mC 双加氧酶进行了分析，包括 ALKBH1 和 TET2，并表明 ALKBH1 是 RNA 中 hmC 和 fC 形成的主要酶。此外，我们使用 5-EC-iCLIP 和基于 ARP 的测序在全转录组范围内对 ALKBH1 修饰位点进行作图，以鉴定各种 tRNA 和 mRNA 中 ALKBH1 依赖性 mC 氧化，并在体外分析 ALKBH1 底物特异性。我们还应用靶向吡啶硼烷介导的测序来测量选定 tRNA 上的 fC 位点。最后，我们表明 tRNA-Leu-CAA 的 wobble 位置上的 fC 在应激下解码 Leu 密码子中起作用。我们的工作为研究 RNA mC 双加氧酶和绘制氧化 mC 修饰提供了强大的化学方法，并揭示了调节 RNA 功能的新型表观转录组途径的存在。
