YTHDC1 cooperates with the THO complex to prevent RNA damage-induced DNA breaks

Certain environmental toxins are nucleic acid damaging agents, as are many chemotherapeutics used for cancer therapy. These agents induce various adducts in DNA as well as RNA. Indeed, most of the nucleic acid adducts (>90%) formed due to these chemicals, such as alkylating agents, occur in RNA. However, compared to the well-studied mechanisms for DNA alkylation repair, the biological consequences of RNA damage are largely unexplored. Here, we demonstrate that RNA damage can directly result in loss of genome integrity. Specifically, we show that a human YTH domain-containing protein, YTHDC1, regulates alkylation damage responses in association with the THO complex (THOC). In addition to its established binding to N6-methyladenosine (m6A)-containing RNAs, YTHDC1 binds to N1-methyladenosine (m1A)-containing RNAs upon alkylation. In the absence of YTHDC1, alkylation damage results in increased alkylation damage sensitivity and DNA breaks. Such phenotypes are fully attributable to RNA damage, since an RNA-specific dealkylase can rescue these phenotypes. These RNA damage-induced DNA breaks (RDIBs) depend on R-loop formation and the processing is mediated by the factors involved in the transcription-coupled nucleotide excision repair. Strikingly, in the absence of YTHDC1 or THOC, an RNA m1A methyltransferase targeted to the nucleus is sufficient to induce DNA breaks. Our results uncover a unique role of YTHDC1-THOC in base damage responses by preventing RDIBs, providing definitive evidence for how damaged RNAs can impact genomic integrity. Overall design: For CLIP-seq, Flag-YTHDC1 expressing HeLa cells were non-treated or treated with alkylating agent MMS for 1 h, then processed with CLIP experiment. The purified RNAs were subjected to library preparation and sequencing. For m1A-DIP-seq, genomic DNAs extracted from control or YTHDC1 KD HeLa cells (treated with MMS) were subjected to m1A immunoprecipitation with m1A antibody. The purified DNAs were processed with library preparation and sequencing. For MapR, MMS-treated control and YTHDC1 KD HeLa cells were harvested for MapR assay incubating with GST-Mnase or GST-Mnase-dRNH. The purified DNAs were processed with library preparation and sequencing.