Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins. [Display omitted] • Alkylated RNA, but not DNA, recruits ASCC-ALKBH3 in an RNF113A-dependent manner • This alkylation repair pathway suppresses transcription and R-loop accumulation • Alkylated pre-mRNA activates RNF113A E3 ligase activity in vitro Tsao et al. demonstrate that RNA and not DNA alkylation is necessary and sufficient to recruit the ASCC-ALKBH3 damage repair complex. The ASCC pathway suppresses transcription and R-loop accumulation in response to alkylation stress. This E3 ubiquitin ligase activity of RNF113A, which recruits this complex, is induced by alkylated RNA. [ABSTRACT FROM AUTHOR]