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Updated: Feb 24, 2026

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU
Published on: August 22, 2019
Osmium-Mediated Transformation of 4-Thiouridine to Cytidine as Key To Study RNA Dynamics by Sequencing
Christian Riml1, Thomas Amort2, Dietmar Rieder3
1Institute of Organic Chemistry and Center for Molecular Biosciences (CMBI), Leopold-Franzens University, Innrain 80-82, 6020, Innsbruck, Austria.
Abstract:
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 OsO4 -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.
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