Trypanosoma brucei ribonuclease H2A is an essential enzyme that resolves R-loops associated with transcription initiation and antigenic variation

In every cell ribonucleotides inserted into DNA genomes represent a threat to the stability and transmission of the genetic material. Two types of Ribonuclease H (RNase H) enzyme are found in most organisms and play key roles tackling ribonucleotides in DNA, either through excision, when the ribonucleotides form part of the DNA strand, or by hydrolysing RNA when it base-pairs with DNA, in structures termed R-loops. Surprisingly, whereas loss of either RNase H1 or RNase H2 is embryonically lethal in mammals, yeast can prosper in the absence of both enzymes. In the protozoan parasite Trypanosoma brucei, loss of RNase H1 is also tolerated but no work has examined the function of RNase H2. Here we show that loss of the catalytic subunit of T. brucei RNase H2 (TbRH2A) leads to growth and cell cycle arrest, which is concomitant with pronounced accumulation of damage in the parasite cell nucleus. Genome-wide mapping reveals that TbRH2A loss results in the combined accumulation of DNA damage and loss of R-loops specifically at sites of RNA polymerase (Pol) II transcription initiation, revealing a novel and critical role for RNase H2. In addition, we detail differential gene expression of both RNA Pol I and II transcribed genes after TbRH2A loss, including patterns that may relate to cytosolic DNA accumulation in humans with autoimmunity caused by RNase H2 mutation. Finally, we show that TbRH2A loss causes R-loop and DNA damage accumulation in telomeric RNA Pol I transcription sites used to express variant surface glycoprotein, undermining immune evasion by antigenic variation. Thus, we demonstrate a separation of function between the two nuclear T. brucei RNase H enzymes during RNA Pol II transcription, but overlap in function during RNA Pol I-mediated gene expression in a critical strategy for parasite-host interaction.

在所有细胞中，插入DNA基因组的核糖核苷酸均会对遗传物质的稳定性与传递构成威胁。多数生物体中存在两类核糖核酸酶H（Ribonuclease H，RNase H），它们在清除DNA中的核糖核苷酸过程中发挥关键作用：当核糖核苷酸作为DNA链的组成部分时通过切除作用将其移除，或是水解与DNA形成碱基配对的RNA（即被称为R环的结构）。令人意外的是，尽管在哺乳动物中单独缺失RNase H1或RNase H2便会导致胚胎致死，但酵母在同时缺失这两种酶的情况下仍可正常存活。在原生动物寄生虫布氏锥虫（Trypanosoma brucei）中，缺失RNase H1同样不会影响其存活，但目前尚无研究探讨RNase H2的功能。本研究显示，布氏锥虫RNase H2的催化亚基（TbRH2A）缺失会导致寄生虫生长受阻与细胞周期停滞，同时伴随寄生虫细胞核内显著的DNA损伤积累。全基因组定位分析表明，TbRH2A缺失会特异地在RNA聚合酶（Pol）II的转录起始位点同时造成DNA损伤积累与R环丢失，这揭示了RNase H2此前未被发现的关键功能。此外，我们详细分析了TbRH2A缺失后，RNA聚合酶I（Pol I）与RNA聚合酶II所转录基因的差异表达情况，其中部分表达模式可能与RNase H2突变引发自身免疫病患者体内的胞质DNA积累相关。最后，我们发现TbRH2A缺失会在用于表达可变表面糖蛋白的端粒RNA聚合酶I转录位点造成R环与DNA损伤积累，进而通过抗原变异破坏寄生虫的免疫逃逸能力。综上，本研究证明，布氏锥虫的两种细胞核RNase H酶在RNA聚合酶II转录过程中功能存在分化，但在RNA聚合酶I介导的基因表达过程中功能存在重叠，而这一过程正是寄生虫与宿主互作的关键策略。