Fig. 1: IWS1 depletion leads to decreased RNA synthesis.
From: IWS1 positions downstream DNA to globally stimulate Pol II elongation
a Schematic representation of dTAG insertion into IWS1 gene using CRISPR/Cas9. Created in BioRender. Zheenbekova, A. (2025) https://BioRender.com/mjvatf1. b Western blots of IWS1 (anti-HA) in lysates of dTAG-IWS1 cells treated with dTAG7 for the indicated time. U1 snRNP 70 is a loading control. The experiment was repeated two times from independent biological replicates. c Boxplots showing fold-changes (dIWS1/control) in RNA synthesis (TT-seq) in expressed genes (All, n = 10,147) and individual gene biotypes (protein coding, n = 9089; lncRNA, n = 615; snRNA, n = 25; snoRNA, n = 41; miRNA, n = 145) after 1 h depletion of IWS1. The thickened line represents the median and the hinges represent the first and third quartiles. The notches stretch to 1.58-times the interquartile range, divided by the square root of the sample size, approximating a 95% confidence interval. Whiskers extend to the maximum and minimum values within 1.5-times the interquartile range from the hinge, outliers are not shown. p-values were determined using one-sample two-sided Wilcoxon test (mu = 0), and shown as n.s = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All: p < 2.2 × 10−16, protein-coding: p < 2.2 × 10−16, lncRNA: p < 2.2 × 10−16, snRNA: p = 0.2411, snoRNA: p = 0.007296, miRNA: p = 2.116 × 10−9. d MA plot of TT-seq data after 1 h depletion of IWS1 showing differential gene expression (n = 10,147). Significantly differentially expressed genes are shown in green and other genes are shown in gray. e Metagene analysis of mean TT-seq coverage in expressed genes (n = 10,147) in 1 h control- or dTAG7-treated (dIWS1) cells. The scaled metagene profiles are aligned at the transcript start (TSS) and transcript end site (TES). The y-axis represents the bootstrapped mean of log2-transformed normalized TT-seq coverage and the shaded areas around the mean indicate 95% confidence intervals of the mean. The gene region is shaded in gray. f As in e), but split into gene length quartiles: shortest (<10 kb, n = 2537), short (10–26.5 kb, n = 2537), long (26.5–65 kb, n = 2536), and longest (> 65 kb, n = 2537) genes.
