Fig. 4: DHX9 SUMOylation suppresses R-loop accumulation and the impairment of transcription termination.

a HeLa cells expressing DHX9^K120R displayed an accumulation of R-loops. HeLa cells with DHX9 depletion by siRNA were transfected with the empty vector, SFB-DHX9^WT, or SFB-DHX9^K120R. DNA/RNA hybrids isolated from nuclear extracts were treated with or without RNH and analyzed using a slot blot assay with the S9.6 antibody. The relative S9.6 signal was quantified (mean ± SEM of three independent experiments). Top: representative blots; Bottom: quantification of relative S9.6 signal from n = 3 biological replicates analyzed by two-sided t test. b Workflow of nucleic acids extraction and the RNH digestion, followed by DRIP assay. c, d HeLa cells treated with control or DHX9 siRNA were transfected with SFB-DHX9^WT, SFB-DHX9^K120R, or the empty vector. DRIP using the S9.6 antibody was analyzed by RT-qPCR for the abundance of R-loops over the polyA-proximal regions of β-actin and γ-actin genes, respectively. Values are normalized to intron 1 (in1) (mean ± SEM of three independent experiments analyzed by two-sided t test). e, f Ablating K120 SUMOylation resulted in transcription termination deficiency. HeLa cells treated with control or DHX9 siRNA were transfected with SFB-DHX9^WT, SFB-DHX9^K120R, or the empty vector. Extracted nucleic acids were analyzed by RT-qPCR for readthrough transcription of β-actin and γ-actin genes, respectively. Data of three biological individual experiments. Values are normalized to β-actin intron 3 (in3) and γ-actin in1 and presented as mean ± SEM (two-sided t test). Source data are provided as a Source Data file.