FICC-Seq: a method for enzyme-specified profiling of methyl-5-uridine in cellular RNA.
Cell Survival
/ drug effects
Deoxyribonucleases
/ chemistry
Fluorouracil
/ pharmacology
HEK293 Cells
High-Throughput Nucleotide Sequencing
/ methods
Humans
RNA
/ chemistry
RNA, Transfer
Saccharomyces cerevisiae
/ genetics
Saccharomyces cerevisiae Proteins
/ chemistry
Uridine
/ chemistry
Yeasts
/ genetics
tRNA Methyltransferases
/ chemistry
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
04 11 2019
04 11 2019
Historique:
accepted:
17
07
2019
revised:
03
07
2019
received:
20
03
2019
pubmed:
31
7
2019
medline:
18
12
2019
entrez:
31
7
2019
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 31361898
pii: 5541094
doi: 10.1093/nar/gkz658
pmc: PMC6821191
doi:
Substances chimiques
Saccharomyces cerevisiae Proteins
0
RNA
63231-63-0
RNA, Transfer
9014-25-9
tRNA Methyltransferases
EC 2.1.1.-
Deoxyribonucleases
EC 3.1.-
TRM2 protein, S cerevisiae
EC 3.1.-
Fluorouracil
U3P01618RT
Uridine
WHI7HQ7H85
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e113Subventions
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/N000749/1
Pays : United Kingdom
Informations de copyright
© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
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