Fig. 1

RBM22-mediated repression of RNAPII pause release at many genes. a Examples of total RNAPII (POLR2A), Ser5P RNAPII (POLR2A-S5P), Ser2P RNAPII (POLR2A-S2P), and RBM22 occupancy measured by ChIP-seq as well as POLR2G occupancy and gene transcription levels measured by GRO-seq at two representative protein-coding genes (SGTA and RPL13) in control (siControl) and RBM22 knockdown (siRBM22) HepG2 cells. b Heatmap and metagene analysis showing the ChIP-seq signal for POLR2A (total; indigo), POLR2A-S5P (purple), POLR2A-S2P (green), and RBM22 (orange) at all human protein-coding genes in HepG2 cells, rank ordered by gene expression. Color-scaled intensities are in units of cpm. c Heatmap and metagene analysis showing the ChIP-seq signal for POLR2G at genes with POLR2G binding at promoters in control and RBM22 knockdown HepG2 cells. Rows are sorted by decreasing POLR2G occupancy in the region between 2 kb upstream of TSS to TES under control condition. For the subtraction of heatmaps, the color bars depict the subtracted values of siRBM22 minus siControl. d RNAPII PRR distribution in control or RBM22 knockdown HepG2 cells, showing increased pause release at many genes after RBM22 knockdown. Higher PRR values indicate a higher degree of pause release. The p value was determined using the Kolmogorov–Smirnov test. e Boxplot showing the fold change (FC) of PRR (POLR2G ChIP-seq) at genes with different degrees of pausing in response to RBM22 depletion. The 9065 genes were equally divided into four groups based on the PRR of RNAPII in the control condition. f Western blot showing the protein abundance of RBM22 in wild-type (WT) cells or in mAID-RBM22 HepG2 cells upon the addition of 5-Ph-IAA for the indicated times. β-actin was used as the loading control. The 5-Ph-IAA is an auxin used for degradation. g POLR2G ChIP-qPCR quantification of RNAPII pause release at four representative protein-coding genes before (without 5-Ph-IAA) or after (with 5-Ph-IAA) RBM22 degradation in mAID-RBM22 cells. The pause release ratio in untreated cells (without 5-Ph-IAA) was set to 1. The p values are determined using the two-tailed unpaired t-test (*p ≤ 0.05; **p ≤ 0.01; ns, not significant). h Metagene analysis showing the elevated GRO-seq signals at protein-coding genes in RBM22 knockdown HepG2 cells. i Metagene analysis of antisense transcription, detected by GRO-seq, at TSS in control and RBM22 knockdown HepG2 cells. j Venn diagram showing the protein-coding genes with both PRR changes in the sense direction and transcriptional changes in the antisense direction. The p value was determined using the hypergeometric test