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

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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, Zuber, Johannes, Ameres, Stefan L.·2017·Nature Methods

DOI 10.1101/177642PMC PMC5712218

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

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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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“Note, that the ability to determine de novo synthesized transcripts will depend on (1) the s(4)U uptake kinetics of the chosen cellular system, (2) the overall transcriptional activity of the cell type and (3) the library sequencing depth.”
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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.
ORCID: Neumann, Tobias
Bhat, Pooja
Burkard, Thomas R.
von Haeseler, Arndt
Zuber, Johannes
Ameres, Stefan L.
Venue: Nature Methods
Publisher: openRxiv
Published: 2017-08-17
Volume: 14
Issue: 12
Pages: 1198-1204
ISSN: 1548-7091 (ISSN-L)
DOI: 10.1101/177642
PMC: PMC5712218
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: green
doi.org
Licence: www.biorxiv.org/about/FAQ#license (vor)
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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