Shen Wen, Sun Hong, De Hoyos Cheryl L, Bailey Jeffrey K, Liang Xue-Hai, Crooke Stanley T
Department of Core Antisense Research, Ionis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, CA 92010, USA.
Nucleic Acids Res. 2017 Oct 13;45(18):10672-10692. doi: 10.1093/nar/gkx710.
An R-loop is a DNA:RNA hybrid formed during transcription when a DNA duplex is invaded by a nascent RNA transcript. R-loops accumulate in nucleoli during RNA polymerase I (RNAP I) transcription. Here, we report that mammalian RNase H1 enriches in nucleoli and co-localizes with R-loops in cultured human cells. Co-migration of RNase H1 and R-loops from nucleoli to perinucleolar ring structures was observed upon inhibition of RNAP I transcription. Treatment with camptothecin which transiently stabilized nucleolar R-loops recruited RNase H1 to the nucleoli. It has been reported that the absence of Topoisomerase and RNase H activity in Escherichia coli or Saccharomyces cerevisiae caused R-loop accumulation along rDNA. We found that the distribution of RNase H1 and Top1 along rDNA coincided at sites where R-loops accumulated in mammalian cells. Loss of either RNase H1 or Top1 caused R-loop accumulation, and the accumulation of R-loops was exacerbated when both proteins were depleted. Importantly, we observed that protein levels of Top1 were negatively correlated with the abundance of RNase H1. We conclude that Top1 and RNase H1 are partially functionally redundant in mammalian cells to suppress RNAP I transcription-associate R-loops.
R环是转录过程中DNA双链被新生RNA转录本侵入时形成的DNA:RNA杂交体。在RNA聚合酶I（RNAP I）转录过程中，R环在核仁中积累。在此，我们报告哺乳动物核糖核酸酶H1在核仁中富集，并在培养的人类细胞中与R环共定位。在抑制RNAP I转录后，观察到核糖核酸酶H1和R环从核仁向核仁周围环状结构的共迁移。用喜树碱处理可短暂稳定核仁R环，从而将核糖核酸酶H1招募到核仁中。据报道，大肠杆菌或酿酒酵母中拓扑异构酶和核糖核酸酶H活性的缺失会导致R环沿核糖体DNA（rDNA）积累。我们发现，在哺乳动物细胞中，核糖核酸酶H1和拓扑异构酶1（Top1）沿rDNA的分布在R环积累的位点重合。核糖核酸酶H1或Top1的缺失都会导致R环积累，而当这两种蛋白都缺失时，R环的积累会加剧。重要的是，我们观察到Top1的蛋白水平与核糖核酸酶H1的丰度呈负相关。我们得出结论，在哺乳动物细胞中，Top1和核糖核酸酶H1在抑制RNAP I转录相关R环方面存在部分功能冗余。
