PreprintCited by 627Single-cell and spatial transcriptomicsRNA Research and SplicingRNA and protein synthesis mechanisms

Thiol-linked alkylation for the metabolic sequencing of RNA

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Herzog, Veronika A., Reichholf, Brian, Neumann, Tobias, Rescheneder, Philipp, Bhat, Pooja, Burkard, Thomas R., Wlotzka, Wiebke, von Haeseler, Arndt +2 more·2017·Nature Methods

Preprint — published as 10.1038/nmeth.4435 · Paper page

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 epitranscriptomics-sequencing technology that uncovers 4-thiouridine (s 4 U)-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 enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. When applied to mouse embryonic stem cells, SLAM-seq provides global and transcript-specific insights into pluripotency-associated gene expression. We validated the method by showing that the RNA-polymerase II-dependent transcriptional output scales 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 N 6 -methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner. One Sentence Summary: Chemical nucleotide-analog derivatization provides global insights into transcriptional and post-transcriptional gene regulation

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Authors
Herzog, V. A., Reichholf, B., Neumann, T., Rescheneder, P., Bhat, P., Burkard, T. R., Wlotzka, W., von Haeseler, A., Zuber, J., & Ameres, S. L.
Affiliations
  • Institute of Molecular Biotechnology (AT)
  • Research Institute of Molecular Pathology (AT)
  • Max Perutz Labs (AT)
Corresponding author
Ameres, Stefan L.
Venue
Nature Methods
Publisher
openRxiv
Published
2017-08-17
Volume
14
Issue
12
Pages
1198-1204
ISSN
1548-7091 (ISSN-L)
OpenAlex
W2759506905
Cited by
622
References
66
Citations by year
2026: 80 · 2025: 79 · 2024: 117 · 2023: 84 · 2022: 71 · 2021: 73 · 2020: 52 · 2019: 44 · 2018: 21 · 2017: 1
FWCI
13.19
Type
journal article
Language
en
Open access
Locations
  • Nature Methods (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • Österreichischen Akademie der Wissenschaften
  • Austrian Science Fund (W12-7-B09, Y-733-B22 START, F4322)
  • Wirtschaftsagentur Wien
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
Keywords
SLAM-seq, RNA turnover, RNA metabolism, RNA polymerase II transcription, gene expression dynamics, microRNA, N6-methyladenosine, post-transcriptional regulation, mouse embryonic stem cells, enhancer activity
MeSH terms
Alkylation, RNA, RNA Polymerase II, RNA Processing, Post-Transcriptional, Sulfhydryl Compounds, Thiouridine, Gene Expression Profiling, Gene Regulatory Networks, Embryonic Stem Cells, High-Throughput Screening Assays, High-Throughput Nucleotide Sequencing

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