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. Researchers at the Institute of Molecular Biotechnology, Vienna have developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry- based epitranscriptomics-sequencing technology that uncovers 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 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. Te researchers validated the method by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and they 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.
Thiol-linked alkylation for the metabolic sequencing of RNA (SLAM-seq)
(a) Workflow of SLAM-seq. Working time for alkylation and Quant-seq library preparation are indicated. (b) Representative genome browser screen shot for three independent mRNA libraries generated from total RNA of mES cells, prepared using standard mRNA sequencing (top panel), Cap-seq (middle panel) and mRNA 3´ end sequencing (bottom panel; RPM, reads per million,). (c) Conversion rates in 3´ UTR-mapping reads of Quant-seq libraries, prepared from mES cells before (no s4U) and after metabolic labeling for 24 h using 100 μM s4U (+s4U). Dashed line represents expected background sequencing error rate. Median conversion rate across the indicated number of transcripts (n) is indicated. P-value (Mann-Whitney test) is indicated. (d) Relative coverage across transcripts for mRNA-seq and Quant-seq. T>C conversion rate (Conv.) distributes evenly within Quant-seq-covered areas.