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Nat Chem Biol. 2017 May;13(5):501-507. doi: 10.1038/nchembio.2317. Epub 2017 Mar 6.

Total RNA-seq to identify pharmacological effects on specific stages of mRNA synthesis.

Author information

1
Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
2
Laboratory of Systems Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
3
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

Abstract

Pharmacological perturbation is a powerful tool for understanding mRNA synthesis, but identification of the specific steps of this multi-step process that are targeted by small molecules remains challenging. Here we applied strand-specific total RNA sequencing (RNA-seq) to identify and distinguish specific pharmacological effects on transcription and pre-mRNA processing in human cells. We found unexpectedly that the natural product isoginkgetin, previously described as a splicing inhibitor, inhibits transcription elongation. Compared to well-characterized elongation inhibitors that target CDK9, isoginkgetin caused RNA polymerase accumulation within a broader promoter-proximal band, indicating that elongation inhibition by isoginkgetin occurs after release from promoter-proximal pause. RNA-seq distinguished isoginkgetin and CDK9 inhibitors from topoisomerase I inhibition, which alters elongation across gene bodies. We were able to detect these and other specific defects in mRNA synthesis at low sequencing depth using simple metagene-based metrics. These metrics now enable total-RNA-seq-based screening for high-throughput identification of pharmacological effects on individual stages of mRNA synthesis.

PMID:
28263964
PMCID:
PMC5688950
DOI:
10.1038/nchembio.2317
[Indexed for MEDLINE]
Free PMC Article

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