Thiol-linked alkylation of RNA to assess expression dynamics.
Nat Methods, 2017/12;14(12):1198-1204.
Herzog VA[1], Reichholf B[1], Neumann T[2], Rescheneder P[3], Bhat P[1], Burkard TR[1], Wlotzka W[1], von Haeseler A[3], Zuber J[2], Ameres SL[1]
Affiliations
PMID: 28945705DOI: 10.1038/nmeth.4435
Impact factor: 47.99
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.
MeSH terms
Alkylation; Embryonic Stem Cells; Gene Expression Profiling; Gene Regulatory Networks; High-Throughput Nucleotide Sequencing; High-Throughput Screening Assays; RNA; RNA Polymerase II; RNA Processing, Post-Transcriptional; Sulfhydryl Compounds; Thiouridine
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