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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.

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


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.

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