RNA/DNA hybrid metabolism and replication stress in cancer

We previously showed that oncogene H-RASV12 causes replication-transcription conflicts by. upregulating components of the general transcription machinery and strongly stimulating RNA synthesis. As a result of activating transcription, H-RASV12 also increases R-loop occupancy across the genome. To identify factors that may help cancer cells to tolerate oncogene-induced R-loops, we screened a panel of proteins that remove R-loops or prevent R-loop formation for expression changes in response to H-RASV12 expression.

RNase H2B displayed the most dramatic change, even compared to RNase H1 and other RNase H2 subunits. RNase H2B expression changed both on the protein and mRNA levels. We further showed that treatment with replication-stress inducing drugs such as hydroxyurea also specifically increased RNase H2B levels, and this may depend on the activity of checkpoint kinases.

RNase H2B is a non-catalytic subunit of RNase H2, which removes both misincorporated ribonucleotides and R-loops from DNA. RNase H2B contains a PIP-box PCNA interaction motif and is responsible for nuclear localisation and interaction of RNase H2 with the DNA replication and repair machinery. We show that RNase H2 subunits are differentially expressed in several cancers. In colorectal cancer (CRC) tissues and cell lines, about 20% of cases have high RNase H2B expression, which correlates with decreased disease-free survival. We have generated RNase H2B- overexpressing CRC cells lines and show that RNase H2B overexpression reduces replication fork stalling and increases cell survival in response to replication stress.

Our data suggest that high levels of RNase H2B may allow cells to tolerate increased ribonucleotide incorporation and R-loop formation during oncogene- and drug-induced replication stress. Our results shed new light on the functions of individual RNase H2 subunits and suggest that increased RNase H2B levels in subsets of cancers may alter RNase H2 activity and promote replication stress resistance.