UAP56/DDX39b is a major co-transcriptional RNA-DNA helicase that unwinds harmful R loops genome-wide

Non-scheduled R loops represent a major source of DNA damage and replication stress. Cells have different ways to prevent R loop accumulation. One mechanism relies on the conserved THO complex in association with co-transcriptional RNA processing factors including the RNA-dependent ATPase UAP56/DDX39B and histone modifiers such as the SIN3 deacetylase in humans. We investigated the function of UAP56/DDX39B in R loop removal. We show that UAP56 depletion causes R loop accumulation, R loop-mediated genome instability and replication fork stalling. We demonstrate an RNA-DNA helicase activity in UAP56 and that its overexpression suppresses R loops and genome instability induced by depleting 5 different unrelated factors. UAP56/DDX39B localizes to active chromatin and prevents the accumulation of RNA-DNA hybrids over the entire genome. We propose that, in addition to its RNA processing role, UAP56/DDX39B is a key helicase required to eliminate harmful co-transcriptional RNA structures that otherwise would block transcription and replication. DNA-RNA immunoprecipitation (DRIP-seq) was performed on untreated or RNase H-treated K562 control cells. DNA-RNA immunoprecipitation strand-specific (DRIPc-seq) were performed on two siC control and siUAP56 K562 cell lines. ChIP-seq and RNA-seq were performed on two K562 control cells.

非计划性R环（non-scheduled R loops）是DNA损伤与复制应激的主要来源。细胞拥有多种抑制R环积累的机制。其中一类机制依赖于保守的THO复合物（THO complex），该复合物可与共转录RNA加工因子结合，包括依赖RNA的ATP酶UAP56/DDX39B以及人类体内的SIN3脱乙酰酶等组蛋白修饰因子。本研究探究了UAP56/DDX39B在R环清除过程中的功能。实验结果表明，敲低UAP56会引发R环积累、R环介导的基因组不稳定性以及复制叉停滞。我们证实UAP56具备RNA-DNA解旋酶活性，且过表达UAP56可抑制由5种不同无关因子敲低所诱导的R环积累与基因组不稳定性。UAP56/DDX39B定位于活性染色质，能够阻止全基因组范围内RNA-DNA杂交体的积累。我们提出，除了在RNA加工过程中发挥作用外，UAP56/DDX39B还是一种关键解旋酶，其对于清除有害的共转录RNA结构至关重要——这类结构若未被清除，将会阻断转录与复制进程。本研究对未处理或经核糖核酸酶H（RNase H）处理的K562对照细胞开展了DNA-RNA免疫沉淀测序（DNA-RNA immunoprecipitation sequencing, DRIP-seq）；对两组siC对照K562细胞系以及siUAP56处理的K562细胞系开展了链特异性DNA-RNA免疫沉淀测序（strand-specific DNA-RNA immunoprecipitation sequencing, DRIPc-seq）；同时对两组K562对照细胞开展了染色质免疫沉淀测序（ChIP-seq）与RNA测序（RNA-seq）。