PARP dependent recruitment of RNA methylated at 8-adenosine is linked to base excision repair mechanism


 Methylation of RNAs, especially 6-methyladenosine (m6A)-modified RNAs, plays a specific role in DNA damage response (DDR). Here, we observed that 8-methyladenosine (m8A)-modified RNA is recruited to UVA-microirradiated chromatin, which was reduced by inhibiting both DNA methylation and histone acetylation, especially in later phases of DDR. Most importantly, clinically used PARP inhibitor (PARPi), olaparib, prevents both m8A and m6A RNA accumulation at microirradiated chromatin. Testing the effect of PARPi on the efficiency of BER, NHEJ, and HR repair pathways, we observed that NHEJ repair proteins are down-regulated after PARP inhibition and recruitment of XRCC1, a factor of BER, to DNA lesions was abolished entirely. Conversely, the PARP inhibitor, olaparib, enhanced the genome-wide level of γH2AX that significantly interacted with m8A RNA, similar to DNA. Together, we showed that the recruitment of m6A RNA and m8A RNA to DNA lesions is PARP dependent, similarly as XRCC1 playing a role in the BER mechanism. We found that γH2AX likely stabilizes m8A/m6A RNA-DNA hybrid loops that are formed during PARP-dependent non-canonical m6A/m8A-mediated DNA repair pathway.


Graphical Abstract: (A)
Non-canonical m 6 A/m 8 A-mediated DNA repair pathway is PARP dependent, and (B) γH2AX stabilizes m 8 A/m 6 A RNA-DNA hybrid loops that are formed as a consequence of genome injury.

Introduction
Cells developed sophisticated mechanisms for maintaining genome integrity. These mechanisms are called DNA damage response (DDR). Genome injury can be seen as singlestrand breaks in DNA, recognized according to the type of damage by base excision repair mechanism (BER) or nucleotide excision repair (NER) [1]. Very deleterious are the doublestrand breaks (DSBs), which erroneous repair leads to tumorigenesis. To avoid pathological processes caused by non-physiological repair of DSBs, the following two canonical and mechanistically distinct repair pathways are initiated: Non-homologous end joining (NHEJ), active mainly in the G1 phase of the cell cycle and less erroneous homologous recombination repair (HRR), which needs the entry of the cells into the S and G2 phases of the cell cycle [2]. It is well-known that the NHEJ repair mechanism also works in S/G2 phases, but only when the HRR process fails [3]. An essential factor of NHEJ repair machinery is p53-binding protein 1 (53BP1) that forms a barrier inhibiting DNA-end resection, mediated via activation of the specific protein cascade involving Mre11/Rad50/NBS1 complex and replication protein A (RPA). This protein binds to 3' single-stranded DNA (ssDNA) generated by nucleolytic degradation of the 5' strands. Subsequently, Rad51 is recruited to DNA lesions, and Rad51positive nucleoprotein filaments are created. After that, in the later step of HRR, there is BRCA1 that promotes DNA end resection by recruiting the CtIP protein to DNA lesions [4].
Also, H3K9me3 represents an epigenetic factor of DDR. An appearance of this epigenetic marker at damaged chromatin is linked to the activation of histone acetyltransferase TIP60, which leads to specific histone acetylation that appears in parallel with phosphorylation of histone H2AX [16][17][18][19]. Also, H4K16 acetylation and H2AXK119 ubiquitination appear at DNA lesions, or sirtuin SIRT1 is recruited to I-SceI-induced DSB sites [20]. Similarly, the NuRD complex, consisting of HDAC1 and HDAC2, is recruited to damaged chromatin to deacetylate H3K56 [21]. We recently observed the histone deacetylation process in the case of H3K9, mediated by histone deacetylase HDAC1 that was recruited to UVA-induced DNA lesions [22].
Recently, it was published that also co-transcriptionally modified RNAs patriciate in DNA damage response. It was shown that there is a novel non-canonical repair pathway mediated via methylated RNA on 6-adenosine (m 6 A) [23][24][25]. This RNA modification is regulated via the function of specific "writes", so call methyl transferases METTL3, METTL14, and METTL16, and recognized by "reader" like the YTHDC1 protein that is additionally associated with specific histone modifications (summarized by [26]). Recently, we additionally showed that mainly METTL16 is recruited to UVA-microirradiated chromatin in later steps of DDR, while the levels of METTL3 and METTL14 proteins remain stable at DSB sites [23]. Xiang et al. (2017) [24] published that methylation at the 6th position of adenosine (m 6 A) in RNA appears at DNA lesions immediately after local laser microirradiation, and we documented that this DDR-related event is additionally accompanied by depletion of 2,2,7methylguanosine (m3G/TMG) in RNA. Significantly, in parallel with described changes in epitranscriptome, UV-irradiation decreases the global cellular level of N 1 -methyladenosine (m 1 A) in RNAs [23]. In this case, mostly when m 6 A RNAs disappeared at DNA lesions, the level of m 6 A "eraser", FTO (fat mass and obesity-associated protein; demethylase) remains stable [23]. Above mentioned experimental data documents that the function of m 6 A-specific "writers" and "erasers" is essential for regulating gene expression and physiological DNA damage repair. For instance, it was shown that in the absence of METTL3, there is a delay in repairing UV-induced cyclobutane pyrimidine dimmer (CPDs) and cells are more sensitive to UV light [24,27]. Also, it is well-known that DNA polymerases participate in DNA damage response. For example, DNA polymerase κ (Pol κ), playing a role in nucleotide excision repair (NER), works in parallel with the catalytic activity of METTL3 that is recruited to damaged chromatin. Xiang et al. (2017) [24] showed that exogenous overexpression of DNA Pol κ rescues the defect of CPDs elimination appearing in METTL-depleted cells. This observation suggests that the fast recruitment of DNA Pol κ to the damage site is potentially due to m 6 A deposition at UV-irradiated genomic regions [27]. Xiang et al. (2017) [24] additionally suggested that METTL3/METTL14 complex (but not METTL3/WTAP complex) in parallel with FTO (but not ALKBH5) serve as the "writers" and the "eraser" of the m 6 A in RNA accumulating at UV-damaged chromatin. Zhang et al. (2020) [28] showed that ATM-mediated phosphorylation at S43 could activate METTL3, and such phosphorylated protein accumulates at DNA lesions, where acts as methyltransferase mediating the m 6 A in RNA. Also, m 6 A "reader", the YTHDC1 protein, is recruited to damaged sites [28]. From this view, the existence of a non-canonical m 6 A-mediated pathway, dependent on the PARP [Poly (ADP-ribose) polymerase], was revealed. This newly-described mechanism is specific for UV-irradiated chromatin but not for ionizing-radiation induced foci (IRIF) [24,26,[29][30][31][32][33][34]. Zhang (2017) [34] suggested that this non-canonical DNA repair pathway works independently on phosphorylation of histone H2AX and is based on the formation of hybrid DNA-RNA loops. It was documented that such DNA-RNA structures appear in the genome as a consequence of DNA damage at transcription sites [35].
Here, we are inspired by the data mentioned above showing novel principles of DNA repair. To continue with the study of the role of RNA modifications in DNA damage response, we addressed whether RNA methylated at 8-adenosine (m 8 A RNA) can recognize UV-induced DNA lesions. It is known that this RNA modification is catalyzed by the SAM (S-Adenosyl methionine)-dependent methyltransferase, Cfr [36]. The studies were performed in bacteria, especially in Escherichia coli, in which the Cfr methyltransferase is responsible for the resistance to five different classes of antibiotics. It was described that the Cfr-mediated modification was determined on nucleotide A2503 of 23S rRNA, and also Cfr can catalyze methylation leading to the formation of 2,8-dimethyladenosine. However, the mutation of single conserved cysteine residues in the SAM motif CxxxCxxC of Cfr abolishes its activity. Here, we have found m 8

Cell cultivation and treatment
The human cervix adenocarcinoma (HeLa) cell line (ATCC® CCL-2TM, ATCC, UK) was cultivated in EMEM (Eagle's Minimum Essential Medium, Merck, Germany) supplemented with 10% fetal calf serum (FCS) and the appropriate antibiotics. To study cell cycle-dependent recruitment of proteins to DNA lesions, we used HeLa-FUCCI cells expressing RFP-Cdt1 in the G1 phase and GFP-geminin in the S/G2/M phases have previously been described in detail [37] (Life Technologies; http://www.lifetechnologies.com). HeLa-FUCCI cells were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% FCS and appropriate antibiotics at 37°C in a humidified atmosphere containing 5% CO2.
The HeLa cells were treated with several different inhibitors listed in Table 1 below. This table summarizes used compounds and their final concentration. Also, it contains the time of treatment duration together with a short description of the main inhibition targets.
The cells were maintained under optimal cultivation conditions in an incubation chamber (EMBL, Germany) at 37°C, and the cell culture hood was supplemented with 5% CO2.
Image acquisition for the induction of DSBs was performed with the following settings: 1024

Immunofluorescence staining
Immunofluorescence was modified, following [22]. The cells were fixed in 4% formaldehyde pixel resolution, 400 Hz, bidirectional mode, and zoom 2. For immunofluorescence analysis, we used Leica Application Suite (LAS X) software, as described above.

FLIM-FRET technique
Fluorescence Lifetime Image (FLIM) Microscopy combined with Förster Resonance Energy Transfer (FRET) was performed following [42]. Using this method, we studied the interactions between XRCC1 protein (donor) and m properties; for example, the higher extinction coefficient (EC) leads to absorbing a more significant amount of light [43]. The specific characteristics of fluorophores used in our FLIM-FRET experiments were adopted from the webpage https://www.fpbase.org/fret/ and are summarized in Table 2. (PicoQuant GmbH, Germany), and FRET efficiency was calculated following [44,45]. By the FLIM-FRET technique, we studied up to 40 cell nuclei for each experimental event.

Statistical analysis
For statistical analyses, we used Sigma Plot 14.5 software (Systat Software, Inc., USA). If the data passed the normality test, then the Student's test was applied. If not, the Mann-Whitney Utest was used. The results of the statistical tests were mentioned directly in the figure legend.

The 8-methyladenosine (m 8 A)-modified RNA is recruited to UVA-microirradiated chromatin
We addressed whether m 6 A RNA [22][23][24] and m 8 A RNA are recruited to UV-induced DNA lesions. In this case, we have observed that m 8 A RNAs recognize locally micro-irradiated chromatin immediately after local laser irradiation. However, m 8 A RNAs positivity weakens 20-30 minutes after the UVA-laser cell exposure (Fig. 1Aa, Ba, C). As the next step, we studied if distinct inhibitors of epigenetic processes and DNA repair can change the recruitment properties of m 8 A RNAs at DNA lesions. We analyzed an effect of Suv20h1/2 inhibitor A196 (A196i) affectioning H4K20me2/me3, a key player of the NHEJ repair mechanism [9,46] (Fig. 1Ab, Bb).
Also, we studied the effects of the following compounds: inhibitors of RNA polymerases I and II (Actinomycin D and α-amanitin) (Fig. 1Ac, Bc, Ad, Bd). Additionally, we performed ATM depletion, which could affect DDR processes (Fig. 1Ae, Be), as well as we studied an effect of inhibition of DNA methyltransferase by 5-aza deoxycytidine (Fig. 1Af, Bf), or inhibition of histone deacetylases (HDACs) by Trichostatin A (TSA) or suberoylanilide hydroxamic acid (Vorinostat; syn. SAHA) (Fig. 1Ag, h and Bg, h). Moreover, we inhibited PARP by olaparib ( Fig. 1Ai, Bi). Quantification analyses showed that ATMi, ACT-D, A196i, α-amanitin strengthen the accumulation of m 8 A RNA at locally micro-irradiated chromatin (Fig. 1C, D), while HDAC inhibitors, TSA, and SAHA (inducing hyperacetylation) reduced the level of m 8 A RNAs at locally-induced DNA lesions (Fig. 1C, D). Importantly, PARP inhibitor olaparib completely abrogated m 8 A RNA recruitment to DNA lesions (Fig. 1Ai, Bi and Fig. 1C, D). Recruitment kinetics of m 8 A RNA to DNA lesions also changed over time. The most pronounced accumulation was 0-5 min after micro-irradiation. The significantly reduced m 8 A RNA signal at DNA lesion was 15-30 min post-irradiation, especially when the cells were treated by 5-AzaC, TSA, or SAHA (Fig. 1C).
To the above-mentioned phenomenon, we additionally observed that the recruitment of In parallel with m 8 A RNA at locally-induced DNA lesions (Fig. 2 RNA to locally-induced DNA lesions (Fig. 3).

PARP inhibitor increased γH2AX positivity in the whole cell nuclei, did not change the level of CPDs at locally induced DNA lesions and abolished recruitment of XRCC1 to microirradiated genomic regions.
We found that PARP inhibitor, olaparib, has no potential to change the levels of XPC and CPDs (factors of nucleotide excision repair mechanism, NER) at DNA lesions. In contrast, olaparib potentiates γH2AX positivity in the whole cell nuclei (Fig. 4A, Ba, c, d). Importantly, PARPi prevents accumulation of XRCC1, a factor of base excision repair, BER, to UVA-damaged chromatin (Fig. 34, Bb). Importantly, PARPi causes an increase of γH2AX positive foci per cell, while did not change the number of XRCC1 positive foci (Fig. 4C, D).

PARP inhibitor reduced the level of the 53BP1 protein, 53BP1pS1778 and RIF1 in microirradiated chromatin, while the level of HRR-related proteins was relatively stable
Due to the fact that PARP inhibitor olaparib abrogated recruitment of m 8 A RNA at DNA lesions, we have analyzed how PARPi affects factors of homologous recombination repair (HRR) and non-homologous end joining (NHEJ) repair mechanism. We have observed that factors of HRR were stable after PARP inhibition at locally-induced DNA lesions. This trend was observed in many post-irradiation intervals (Fig. 5Aa-c, Ba-c). An exception was up-regulation of the MDC1 protein in the later step of DDR; 20-30 after microirradiation (Fig. 5Ac, Bc). Interestingly, the number of DNA repair foci induced by microirradiation was significantly increased by olaparib treatment when we studied MDC1-positivity (Fig. 5Cc). However, BRCA1-and RAD51positive foci were not changed when the cells were treated with olaparib (Fig. 5Ca, b).
Besides HRR factors, we have studied proteins involved in the HNEJ repair pathway.
We analyzed recruitment kinetics to microirradiated chromatin for 53BP1, its phosphorylated form, 53BP1pS1778, and the RIF1 protein. We observed protein down-regulation at DNA lesions in all cases studied when the cells were treated by PARPi (Fig. 6Aa-c, Ba-c). Notably, the number of DNA repair foci after microirradiation was not changed by olaparib treatment (Fig. 6Ca-c).

m 8 A RNA interacts identically with DNA and γH2AX
Using FLIM-FRET analysis, we studied a degree of interaction between m8A RNA and XRCC1, a protein that, similarly to m 8 A RNA, was recruited to DNA lesions in normal cells. Still, the treatment by PARP inhibitor abolished the accumulation of both m 8 A RNA and XRCC1 at microirradiated chromatin (Fig. 1Ai, 4A, Bb). In this case, we have observed the interaction properties on the so-called cut-off level that we established at 20% when measuring FLIM-FRET efficiency [47] (Fig. 7a). However, a significant FLIM-FRET efficiency was observed when we measured m 8 A RNA interaction with γH2AX or m 8 A RNA binding to DNA. In these cases, FLIM-FRET efficiency was approximately 30%. Interestingly, UVA-local microirradiation did not change the interaction properties of studied partners (Fig. 7b, c). Due to high FLIM-FRET efficiency for m 8 A RNA and DNA, these data support the existence of m 8 A RNA-DNA hybrids that stabilize the genome after injury in the form of R-loops. Based on our FLIM-FRET data, it seems likely that also γH2AX contributes to this process (see high FLIM-FRET efficiency in Fig.   7b).

Discussion
Yu et al. (2021) [48] showed that the function of the METTL3-METTL14 complex, similarly to m 6 A RNA nuclear reader YTHDC1, contributes to the repair of DNA lesions containing cyclobutene pyrimidine dimers, which are well-characterized elements that appear after UVirradiation. Moreover, N6-methyladenine (N6mA) reduced misincorporation of 8-oxo-guanine (8-oxoG) opposite to N6mA by repair DNA polymerases. When 8-oxoG is incorporated into the opposite site to N6mA, this process inhibits N6mA excision from the template [48]. From this observation, it is evident that METTL3/METTL14 methyltransferases, together with m 6 A RNAs, are required for DNA damage repair mechanisms that are initiated by UV radiation [23,24].

Supplementary Files
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