Pol II distribution and pausing are altered with disruption of cotranscriptional splicing. (A) ChIP-seq signal of Pol II (left), p-Ser2 Pol II (middle), and p-Ser5 Pol II (right) from NALM6 cells treated with DMSO and EHT1610 (5 μM). The average profiles of DMSO and EHT1610 that covered ±2 kb of the gene were plotted. (B) Snapshots of RBM39 loci (IGV browser) with ChIP-seq signals for Pol II, p-Ser2 Pol II, and p-Ser5 Pol II upon EHT1610 treatment in NALM6 cells. (C) ChIP-seq signal of p-Ser2 Pol II (left) and p-Ser5 Pol II (right) from NALM6 cells treated with DMSO and THAL-SNS-032 (500 nM). The average profiles of DMSO and THAL-SNS-032 were plotted and covered ±3kb of the gene. (D) Snapshots of RBM39 loci (IGV browser) with ChIP-seq signals for p-Ser2 Pol II and p-Ser5 Pol II upon THAL-SNS-032 treatment in NALM6 cells. (E) Venn diagram showing overlapped alterative spliced genes (analyzed from Figure 1B) and decreased p-Ser5 Pol II ChIP-seq signal genes upon EHT1610 treatment. (F) Venn diagram showing overlapped alternatively spliced genes (analyzed from Figure 2C) and differential p-Ser5 Pol II ChIP-seq signal genes upon THAL-SNS-032 treatment. (G-J) NALM6 cells were treated with DMSO and EHT1610 (5 μM) for 4 hours and with THAL-SNS-032 (500 nM) for 8 hours followed by PRO-seq. (G) Comparison of the pausing indexes of the overlapping genes in (F). (H) Comparison of pausing indexes of those overlapped genes in (E). (I) Metagene plot showing PRO-seq signal in NAML6 cells treated with DMSO and EHT1610 for those overlapped genes in (E). (J) Snapshot of PRO-seq track on the RBM39 locus in NALM6 cells with DMSO, EHT1610, and THAL-SNS-032 treatment. (+) indicates transcription from left to right; (−) indicates transcription from right to left. The pausing indexes for the RBM39 locus are indicated to the right (n = 2; ∗P < .05).