N6-Adenosine Methylation in RNA and a Reduced m3G/TMG Level in Non-Coding RNAs Appear at Microirradiation-Induced DNA Lesions

Abstract

The DNA damage response is mediated by both DNA repair proteins and epigenetic markers. Here, we observe that N6-methyladenosine (m6A), a mark of the epitranscriptome, was common in RNAs accumulated at UV-damaged chromatin; however, inhibitors of RNA polymerases I and II did not affect the m6A RNA level at the irradiated genomic regions. After genome injury, m6A RNAs either diffused to the damaged chromatin or appeared at the lesions enzymatically. DNA damage did not change the levels of METTL3 and METTL14 methyltransferases. In a subset of irradiated cells, only the METTL16 enzyme, responsible for m6A in non-coding RNAs as well as for splicing regulation, was recruited to microirradiated sites. Importantly, the levels of the studied splicing factors were not changed by UVA light. Overall, if the appearance of m6A RNAs at DNA lesions is regulated enzymatically, this process must be mediated via the coregulatory function of METTL-like enzymes. This event is additionally accompanied by radiation-induced depletion of 2,2,7-methylguanosine (m3G/TMG) in RNA. Moreover, UV-irradiation also decreases the global cellular level of N1-methyladenosine (m1A) in RNAs. Based on these results, we prefer a model in which m6A RNAs rapidly respond to radiation-induced stress and diffuse to the damaged sites. The level of both (m1A) RNAs and m3G/TMG in RNAs is reduced as a consequence of DNA damage, recognized by the nucleotide excision repair mechanism.

Context-Dependent Roles of RNA Modifications in Stress Responses and Diseases

“…METTL16, a U6 snRNA methyltransferase, was also recruited to DNA damage sites at a later time point (20-30 min post-UV) and was found to methylate small nuclear and nucleolar RNAs that were also recruited to DNA damage sites upon UV radiation [58]. m 6 A was only recruited to DNA damage lesions only in the presence of cyclobutane pyrimidine dimers (CPDs), which form in response to UV exposure [58]. Furthermore, in response to UV, m 6 A RNA modifications may utilize the nucleotide excision repair (NER) pathway rather than nonhomologous end-joining (NHEJ), as knockout of two NHEJ-specific enzymes, SUV391H/H2, had no effect on m 6 A recruitment [58].…”

Epigenetics and DNA Repair in Cancer

“…Colocalization of Pol κ with m6A to sites with a high content of cyclobutene pyrimidine dimers (CPDs) corroborate that m6A RNAs have a regulatory role in the NER pathway. Svobodová et al [108] found that m6A RNAs are diffuse to damaged DNA, but a new participant, METTL16, accumulated 20-30 min after induced damage in a subset of irradiated cells. This response was specific to CPDs, as the authors observed that m6A RNAs' accumulation pattern was specific to repair of CPDs' sites, which do not accumulate in other lesions, such as NHEJ.…”

Jack of all trades? The versatility of RNA in DNA double-strand break repair

“…The RNA m 6 A-modification accumulates rapidly at UV laser microirradiated sites, colocalising with the m 6 A writer METTL3–METTL14 complex. More generally, UVC induced m 6 A in a variety of transcripts, indicating a possible dual role of m 6 A in the UV DDR, both at the site of the damage and more globally within the cell [ 133 , 134 ]. m 6 A was found to be required for the recruitment of downstream repair factors, such as Polymerase κ (Pol κ), although DSB repair factors such as BRCA1 and 53BP1 were not dependent on the m 6 A writer METTL3 for recruitment to damage [ 133 ].…”

“…Interestingly, this m 1 A RNA modification can be reversed by ALKBH3, an enzyme with a known role in DNA repair [ 140 ]. While m 1 A has not been shown to be recruited to UV-microirradiation in the same manner as m 6 A [ 134 ] further studies may shed light on a potential role of in the DDR. However, it is important to note that there has been controversy regarding the m 1 A antibody, suggesting m 1 A peaks identified in 5′UTRs are likely the result of antibody cross-reactivity with the m 7 G-cap of RNA [ 143 ].…”