GSE109183
FICC-Seq: A method for enzyme-specified profiling of methyl-5-uridine in cellular RNA
Methyl-5-uridine (m5U) is one the most abundant non-canonical bases present in cellular RNAs, and is formed via catalytic conversion of uridines. In yeast, m5U is known to be present at position 54 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 map the relevant enzymatic targets in cellular RNA. We demonstrate that TRMT2A is responsible for the majority of m5U present in human RNA, and that it ubiquitously 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 demonstration of irreversible human m5U enzyme-RNA crosslinking in vivo following 5-flurouracil exposure is further intriguing, as it suggests a tangible mechanism for a previously suspected RNA-dependent route of 5-fluorouracil-mediated cytotoxicity. Overall design: Examination of novel FICC-Seq method in comparison with iCLIP and miCLIP.