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 researchers from Harvard Medical School applied strand-specific total RNA sequencing (RNA-seq) to identify and distinguish specific pharmacological effects on transcription and pre-mRNA processing in human cells. They 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. The researchers 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.
The topoisomerase inhibitor camptothecin (CPT) decreases pre-mRNA
expression along the bodies of long genes.
(a) CPT treatment of HeLa cells (1 μM for 18 h) leads to a decrease in intron expression at TSS-distal locations detectable by strand-specific total RNA-seq at the human Gpc5 locus. (b) A TSS metaplot reveals decreased intronic expression at TSS-distal locations upon CPT treatment (quantified and statistically assessed in c), normalized to the number of mRNA (exon)-aligned reads at least 10 kb from the TSS. Only the 850 genes longer than 250 kb are included. The inset shows the metaplot for −5 to 10 kb relative to the TSS (c) Density change across long gene bodies metric based on the metaplot in b: the ratio of the sense RNA-seq signal +100 to +150 kb to +200 to +250 kb relative to the TSS, divided by the same ratio in controls. Stars indicate significant increases (P < 0.05) based on a one-tailed t-test with Bonferroni correction. (d) Antisense TSS/sense TSS, TSS/gene body, intron/exon, and PAS-downstream/PAS metrics calculated for CPT treatment; these values are repeated here for reference. None of the CPT effects are statistically significant. CPT data in b–d are means from n = 2 replicates.