Human RNaseH2 subunit upregulation counteracts oncogene- and chemotherapy-induced replication stress

RNaseH2 is a heterotrimeric endoribonuclease that resolves RNA:DNA hybrids and mis-incorporated ribonucleotides, which are implicated in DNA replication stress and cancer development and potential therapeutic targets. Individual RNaseH2 subunit mRNA or protein levels are found elevated in some cancers, but little is known about the mechanisms behind and impact of RNaseH2 subunit upregulation. We report that RNaseH2 subunits are upregulated at the protein level in response to replication stress induced by oncogenes and chemotherapy drugs hydroxyurea, gemcitabine and camptothecin in human cancer and non-cancer cell lines. We show that inducible overexpression of the RNaseH2B subunit increases the levels of the whole active RNaseH2 complex and can counteract drug-induced RNA:DNA hybrid formation. RNaseH2B overexpression reduces replication fork stalling induced by camptothecin and hydroxyurea but can itself increase RNA:DNA hybrid levels and cause replication stress. RNaseH2 subunit levels do not strongly impact survival of chemotherapy drug treatments but may have more subtle effects on genomic instability and cytoplasmic DNA signalling. In contrast, increased RNaseH2 subunit levels in presence of oncogenic HRAS are required to limit not only RAS-induced replication fork stalling but also cell death. Our findings shed new light on the functions of RNaseH2 and suggest that upregulation of RNaseH2 subunits may be an important aspect of replication stress responses in cancer. Overall design: To test whether RNaseH2B overexpression alters gene expression, we performed RNA sequencing on HCT116 cells overexpressing RNaseH2B. Sequencing was peformed on HCT116 cells before and 48 hours after doxycycline-induced RNaseH2B induction. Parental HCT116 cells before and after doxycycline treatment were used as a control. We then performed gene expression profiling analysis using data obtained from RNA-Seq.

核糖核酸酶H2 (RNaseH2) 是一种异三聚体核糖核酸内切酶，可解离RNA:DNA杂交结构与错误掺入的核糖核苷酸，其与DNA复制压力、癌症发生密切相关，同时也是潜在的治疗靶点。
部分癌症中可检测到单个RNaseH2亚基的mRNA或蛋白水平上调，但目前对RNaseH2亚基上调的背后机制及其影响仍知之甚少。
本研究发现，在人类癌症细胞系与非癌症细胞系中，RNaseH2亚基的蛋白水平会在致癌基因及羟基脲、吉西他滨、喜树碱等化疗药物诱导的复制压力下出现上调。
研究表明，诱导性过表达核糖核酸酶H2B (RNaseH2B) 亚基可提升具有活性的完整RNaseH2复合物水平，并能抵消药物诱导的RNA:DNA杂交结构形成。
RNaseH2B过表达可减少喜树碱与羟基脲诱导的复制叉停滞，但自身也会提升RNA:DNA杂交结构水平并引发复制压力。
RNaseH2亚基水平对化疗药物处理后的细胞存活无显著影响，但可能对基因组不稳定性与细胞质DNA信号通路产生更为细微的调控作用。
与之相反，在致癌性HRAS存在的情况下，RNaseH2亚基水平升高不仅是限制RAS诱导的复制叉停滞所必需的，同时也是抑制细胞死亡所必需的。
本研究结果为RNaseH2的功能提供了新的见解，并提示RNaseH2亚基上调可能是癌症中复制压力应答的重要环节。
整体实验设计：为验证RNaseH2B过表达是否会改变基因表达，我们对过表达RNaseH2B的HCT116细胞进行了RNA测序。分别在多西环素诱导RNaseH2B表达前及诱导48小时后，对HCT116细胞进行测序；以未经诱导的亲本HCT116细胞及其多西环素处理组作为对照。随后，我们利用RNA测序获得的数据进行了基因表达谱分析。