RT Journal Article
SR Electronic
T1 Thiol-linked alkylation for the metabolic sequencing of RNA
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 177642
DO 10.1101/177642
A1 Herzog, Veronika A.
A1 Reichholf, Brian
A1 Neumann, Tobias
A1 Rescheneder, Philipp
A1 Bhat, Pooja
A1 Burkard, Thomas R.
A1 Wlotzka, Wiebke
A1 von Haeseler, Arndt
A1 Zuber, Johannes
A1 Ameres, Stefan L.
YR 2017
UL http://biorxiv.org/content/early/2017/08/17/177642.abstract
AB 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 (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. 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 N6-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