Thiol-linked alkylation of RNA to assess expression dynamics
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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 RNA sequencing technology that detects 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 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 N 6-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, 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
- Published
- 2017-09-25
- Volume
- 14
- Issue
- 12
- Pages
- 1198-1204
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- PMC
- PMC5712218
- OpenAlex
- W2759506905
- Cited by
- 622
- References
- 7
- 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
- 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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