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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We thank M. Hemberg and D. Harmin for advice on bioinformatic analyses. This work was funded by R01 MH101528-01. M.S. is also supported by National Science Foundation grant 1349248. S.A.B. is supported by the Harvard Medical School Center of Excellence in Systems Pharmacology NIH grant P50 GM107618 and the Giovanni Armenise-Harvard Foundation. H.M.L. and L.S.C's contributions were funded by NHGRI: R01 HG007173. We thank K. Koide (University of Pittsburgh) for sharing meayamycin.
The authors declare no competing financial interests.
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Boswell, S., Snavely, A., Landry, H. et al. Total RNA-seq to identify pharmacological effects on specific stages of mRNA synthesis. Nat Chem Biol 13, 501–507 (2017). https://doi.org/10.1038/nchembio.2317
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