N1-methyladenosine (mA) is an important post-transcriptional modification in RNA; however, the exact biological role of mA remains to be determined. By employing a quantitative proteomics method, we identified multiple putative protein readers of mA in RNA, including several YTH domain family proteins. We showed that YTHDF1-3 and YTHDC1, but not YTHDC2, could bind directly to mA in RNA. We also found that Trp in YTHDF2, a conserved residue in the hydrophobic pocket of the YTH domain that is necessary for its binding to N-methyladenosine (mA), is required for its recognition of mA. An analysis of previously published data revealed transcriptome-wide colocalization of YTH domain-containing proteins and mA sites in HeLa cells, suggesting that YTH domain-containing proteins can bind to mA in cells. Together, our results uncovered YTH domain-containing proteins as readers for mA in RNA and provided new insight into the functions of mA in RNA biology.
Most mammalian cells in nature are quiescent but actively transcribing mRNA for normal physiological processes; thus, it is important to investigate how endogenous and exogenous DNA damage compromises transcription in cells. Here we described a novel competitive transcription and adduct bypass (CTAB) assay to determine the effects of DNA lesions on the fidelity and efficiency of transcription. Using this strategy, we demonstrated that the oxidatively induced lesions 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG), and methylglyoxal-induced N2-(1-carboxyethyl)-2′-deoxyguanosine (N2-CEdG) strongly inhibited transcription in vitro and in mammalian cells. In addition, cdA and cdG, but not N2-CEdG, induced transcriptional mutagenesis in vitro and in vivo. Furthermore, when located on the template DNA strand, all examined lesions were primarily repaired by transcription-coupled nucleotide excision repair (TC-NER) in mammalian cells. This newly developed CTAB assay should be generally applicable for quantitatively assessing how other DNA lesions impact DNA transcription in vitro and in cells.
Background:The 8,5Ј-cyclopurine-2Ј-deoxynucleosides (cPus) are important types of oxidative DNA damage. Results: cPus exhibit both inhibitory and mutagenic effects on replication; polymerases (Pols) , , and are involved in translesion synthesis of these lesions. Conclusion: Pols , , and cooperatively promote translesion synthesis across cPu lesions. Significance: This work revealed the effects of cPus on DNA replication in mammalian cells.
Background: Because its N terminus adopts an APK motif, DDB2 might be ␣-N-methylated. Results: We examined the nature of DDB2 ␣-N-methylation, the enzyme involved in this methylation and its function in DNA repair. Conclusion: DDB2 could be ␣-N-methylated by NRMT, and this methylation facilitated the recruitment of DDB2 to DNA damage foci. Significance: This work expands the function of protein ␣-N-methylation to DNA repair.
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