FICC-Seq: a method for enzyme-specified profiling of methyl-5-uridine in cellular RNA

被引:49
作者
Carter, Jean-Michel [1 ]
Emmett, Warren [2 ,3 ]
Mozos, Igor R. D. L. [2 ,4 ]
Kotter, Annika [5 ]
Helm, Mark [5 ]
Ule, Jernej [2 ,4 ]
Hussain, Shobbir [1 ]
机构
[1] Univ Bath, Dept Biol & Biochem, Bath BA2 7AY, Avon, England
[2] Francis Crick Inst, 1 Midland Rd, London NW1 1AT, England
[3] UCL, Genet Inst, Gower St, London WC1E 6BT, England
[4] UCL Queen Sq Inst Neurol, Dept Neuromuscular Dis, London WC1N 3BG, England
[5] Johannes Gutenberg Univ Mainz, Inst Pharm & Biochem, Staudinger Weg 5, D-55128 Mainz, Germany
基金
英国生物技术与生命科学研究理事会;
关键词
PSEUDOURIDINE SYNTHASES; SUBSTRATE SELECTIVITY; 5-FLUOROURACIL; IDENTIFICATION; MECHANISM; METHYLTRANSFERASE; CONTRIBUTES; NUCLEOTIDE; DATABASE; REVEALS;
D O I
10.1093/nar/gkz658
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:E113 / +
页数:12
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