Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
The Francis-Crick Institute, 1 Midland Road, London, NW1 1AT, UK.
Nucleic Acids Res. 2019 Nov 4;47(19):e113. doi: 10.1093/nar/gkz658.
Methyl-5-uridine (m5U) is one the most abundant non-canonical bases present in cellular RNA, and in yeast is found at position U54 of tRNAs where modification is catalysed by the methyltransferase Trm2. Although the mammalian enzymes that catalyse m5U formation are yet to be identified via experimental evidence, based on sequence homology to Trm2, two candidates currently exist, TRMT2A and TRMT2B. Here we developed a genome-wide single-nucleotide resolution mapping method, Fluorouracil-Induced-Catalytic-Crosslinking-Sequencing (FICC-Seq), in order to identify the relevant enzymatic targets. We demonstrate that TRMT2A is responsible for the majority of m5U present in human RNA, and that it commonly targets U54 of cytosolic tRNAs. By comparison to current methods, we show that FICC-Seq is a particularly robust method for accurate and reliable detection of relevant enzymatic target sites. Our associated finding of extensive irreversible TRMT2A-tRNA crosslinking in vivo following 5-Fluorouracil exposure is also intriguing, as it suggests a tangible mechanism for a previously suspected RNA-dependent route of Fluorouracil-mediated cytotoxicity.
甲基化-5-尿嘧啶（m5U）是细胞 RNA 中最丰富的非规范碱基之一，在酵母中位于 tRNA 的 U54 位置，由甲基转移酶 Trm2 催化修饰。尽管通过实验证据尚未鉴定出催化 m5U 形成的哺乳动物酶，但基于与 Trm2 的序列同源性，目前存在两个候选物，TRMT2A 和 TRMT2B。在这里，我们开发了一种全基因组单核苷酸分辨率映射方法，即氟尿嘧啶诱导催化交联测序（FICC-Seq），以鉴定相关的酶靶标。我们证明 TRMT2A 负责人类 RNA 中存在的大多数 m5U，并且它通常靶向细胞质 tRNA 的 U54。与当前方法相比，我们表明 FICC-Seq 是一种特别稳健的方法，可用于准确可靠地检测相关酶靶标位点。我们还发现，在 5-氟尿嘧啶暴露后，体内 TRMT2A-tRNA 的广泛不可逆交联是有趣的，因为它为先前怀疑的基于 RNA 的氟尿嘧啶介导细胞毒性的途径提供了一种有形的机制。
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