Osmium-Mediated Transformation of 4-Thiouridine to Cytidine as Key To Study RNA Dynamics by Sequencing

Angew Chem Int Ed Engl. 2017 Oct 16;56(43):13479-13483. doi: 10.1002/anie.201707465. Epub 2017 Sep 18.

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.

Keywords: RNA modification; bioorganic chemistry; cyclins; gene sequencing; metabolic labeling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ammonium Chloride / chemistry
  • Chromatography, Ion Exchange
  • Cytidine / chemistry*
  • HEK293 Cells
  • Humans
  • Osmium / chemistry*
  • RNA / chemistry*
  • RNA / metabolism
  • Sequence Analysis, RNA
  • Spectrometry, Mass, Electrospray Ionization
  • Temperature
  • Thiouridine / chemistry*

Substances

  • Ammonium Chloride
  • Thiouridine
  • Osmium
  • Cytidine
  • RNA