Thiol-linked alkylation of RNA to assess expression dynamics

Thiol-linked alkylation of RNA to assess expression dynamics

Abstract

Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq), an orthogonal-chemistry-based RNA sequencing technology that detects 4-thiouridine (s4U) incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM seq enabled rapid access to RNA-polymerase-II-dependent gene expression dynamics in the context of total RNA. We validated the method in mouse embryonic stem cells by showing that the RNA-polymerase-II-dependent transcriptional output scaled with Oct4/Sox2/Nanog-defined enhancer activity, and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective and scalable manner.

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Authors
  • Herzog, Veronika A.
  • Reichholf, Brian
  • Neumann, Tobias
  • Rescheneder, Philipp
  • Bhat, Pooja
  • Burkard, Thomas R.
  • Wlotzka, Wiebke
  • von Haeseler, Arndt
  • Zuber, Johannes
  • Ameres, Stefan L.
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Shortfacts
Category
Journal Paper
Divisions
Bioinformatics and Computational Biology
Journal or Publication Title
Nature Methods
ISSN
1548-7091
Publisher
Macmillan Publishers Limited, part of Springer Nature
Place of Publication
London N1 9XW United Kingdom
Date
25 September 2017
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