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Osmium‐Mediated Transformation of 4‐Thiouridine to Cytidine as Key To Study RNA Dynamics by Sequencing
Author(s) -
Riml Christian,
Amort Thomas,
Rieder Dietmar,
Gasser Catherina,
Lusser Alexandra,
Micura Ronald
Publication year - 2017
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201707465
Subject(s) - rna , cytidine , biology , computational biology , messenger rna , gene expression , dna microarray , transformation (genetics) , microbiology and biotechnology , gene , biochemistry , enzyme
To understand the functional roles of RNA in the cell, it is essential to elucidate the dynamics of their production, processing and decay. A recent method for assessing mRNA dynamics is metabolic labeling with 4‐thiouridine (4sU), followed by thio‐selective attachment of affinity tags. Detection of labeled transcripts by affinity purification and hybridization to microarrays or by deep sequencing then reveals RNA expression levels. Here, we present a novel sequencing method (TUC‐seq) that eliminates affinity purification and allows for direct assessment of 4sU‐labeled RNA. It employs an OsO 4 ‐mediated transformation to convert 4sU into cytosine. We exemplify the utility of the new method for verification of endogenous 4sU in tRNAs and for the detection of pulse‐labeled mRNA of seven selected genes in mammalian cells to determine the relative abundance of the new transcripts. The results prove TUC‐seq as a straight‐forward and highly versatile method for studies of cellular RNA dynamics.