RESUMO
Physiological and chemically induced modifications to nucleosides are common in both DNA and RNA. Physiological forms of these modifications play critical roles in gene expression, yet aberrant marks, if left unrepaired, may be associated with increased genome instability. Due to the low prevalence of these marks in most samples of interest, a highly sensitive method is needed for their detection and quantitation. High-performance liquid chromatography, coupled to mass spectrometry (HPLC-MS), provides this high degree of sensitivity while also being adaptable to nearly any modified nucleoside of interest and still maintaining exquisite specificity. In this chapter, we demonstrate how to use HPLC-MS to analyze the catalytic activity of a nucleic acid demethylase, to quantify the prevalence of N6-methyladenosine from RNA, and to determine the kinetics of alkylation damage repair.
Assuntos
Nucleosídeos , RNA , Cromatografia Líquida de Alta Pressão/métodos , DNA , Espectrometria de Massas , Nucleosídeos/químicaRESUMO
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.
