title: Total RNA-seq to identify pharmacological effects on specific stages of mRNA synthesis.
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.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.|Laboratory of Systems Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Boswell
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Snavely
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Heather M
authors.initials: HM
authors.lastname: Landry
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: L Stirling
authors.initials: LS
authors.lastname: Churchman
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Jesse M
authors.initials: JM
authors.lastname: Gray
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.|Laboratory of Systems Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Springer
mesh_terms: D044946:Biflavonoids / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D017423:Sequence Analysis, RNA; D061805:Transcription Elongation, Genetic / Q000187:drug effects*
chemical_list: D044946:Biflavonoids; D012333:RNA, Messenger; C452984:isoginkgetin