4.8 Article

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

Journal

NATURE CHEMICAL BIOLOGY
Volume 13, Issue 5, Pages 501-+

Publisher

NATURE PUBLISHING GROUP
DOI: 10.1038/NCHEMBIO.2317

Keywords

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Funding

  1. National Science Foundation [1349248]
  2. Harvard Medical School Center of Excellence in Systems Pharmacology NIH [P50 GM107618]
  3. Giovanni Armenise-Harvard Foundation
  4. [R01 MH101528-01]
  5. [NHGRI: R01 HG007173]
  6. Direct For Biological Sciences
  7. Div Of Molecular and Cellular Bioscience [1349248] Funding Source: National Science Foundation

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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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