N6-methyladenosine (m6A) Interplays with RNA and DNA Damage to Regulate DNA Repair

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N6-methyladenosine (m6A) is the most common modification in mRNA and regulates gene expression. Modulation of m6A profiles is associated with cancer progression and chemotherapeutic drug resistance. However, it remains unknown how m6A is involved in the processes. Understanding the underlying mechanisms is the key to the discovery of new drug targets and biomarkers for cancer therapy and diagnosis. Recent studies have pointed to a new role of m6A in regulating RNA-guided DNA repair suggesting that it can mediate cancer development by interplaying with DNA damage and repair. We recently found that an m6A located next to the stop codon on the transcript of DNA polymerase β (Pol β) was eliminated by oxidative RNA base damage induced by the environmental toxicant, potassium bromate (KBrO3), shifting its deposition to the upstream of the Pol β transcript. We hypothesize that m6A interplays with RNA and DNA base damage to regulate DNA repair. To test this hypothesis, we will pursue two Specific Aims. Aim 1 is to determine if m6A profiles and abundance can be modulated by RNA and DNA base damage in cancer cells. First, we will determine if oxidative RNA and DNA base damage can disrupt the profiles of m6A on the Pol β transcript in cancer cells. This will be tested in HeLa cells treated with KBrO3. We will determine if m6A profiles can be modulated by oxidative RNA base damage, 8-oxoG, and abasic sites (AP sites) on the Pol β transcript using DNA-RNA immunoprecipitation (DRIP)-mediated strand break-mediated RNA modification profiling assay (DRIP-SBRM). We will then determine if the m6A profiles can be altered by DNA base damage, 8-oxodGs, and AP sites on the DNA template of Pol β transcript using the DRIP-mediated strand break-mediated DNA modification profiling assay (DRIP- SBDM). Second, we will determine if RNA and DNA base damage can alter the activity and substrate binding affinity of the m6A writer and eraser, METTL3/METTL14 and FTO. Aim 2 is to determine if m6A can alter the accumulation of RNA base damage to modulate DNA repair in cancer cells. First, we will determine if the profiles of oxidative DNA and RNA base damage on the Pol β transcript can be modified by m6A under METTL3 deficiency in HeLa cells. The effects of m6A on the profiles of oxidative DNA and RNA base damage induced by KBrO3 will be determined using HeLa cells with or without METTL3 gene knockdown. The profiles of m6A and oxidative base damage on the Pol β transcript and its template DNA will be correlated. Second, we will determine if the activity and substrate binding affinity of the key BER enzymes can be modulated in an m6A and RNA damage location-dependent manner. The study will prove the new concept that epitranscriptomic modifications interplay with RNA and DNA base damage to regulate DNA repair in cancer cells. The results will reveal a novel paradigm for m6A to interplay with oxidative RNA and DNA damage to modulate DNA repair that underlies cancer progression. Thus, the study will facilitate the discovery of new targets for RNA-based treatment, diagnosis, and prevention of environmentally-induced cancer.