TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution

Abstract

R-loops are three-stranded, RNA–DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.

Thrap3 promotes R-loop resolution via interaction with methylated DDX5

“…For example, RNA m6A (methylation of an A base), which is important for many RNA metabolic processes, is highly enriched at sites of UV irradiation-induced DNA damage for DNA polymerase kappa (POLK) recruitment 30 . In addition, TonEBP, a pleiotropic transcription factor, plays a role in the DNA damage response through interaction with various proteins related to PCNA polyubiquitination or m6A RNA methylation 27 , 28 . According to our unpublished data, Thrap3 binds to TonEBP and induces PCNA ubiquitination under conditions of DNA damage.…”

“…Proteins that regulate gene transcription also have functions in other DNA processes, such as DNA replication and repair [24][25][26][27][28][29] . For example, RNA m6A (methylation of an A base), which is important for many RNA metabolic processes, is highly enriched at sites of UV irradiation-induced DNA damage for DNA polymerase kappa (POLK) recruitment 30 .…”

N6-Methyladenosine, DNA Repair, and Genome Stability

“…TonEBP binds to R-loops and recruits METTL3 that generates m 6 A on the RNA strand of the R-loop. TonEBP then recruits RNaseH1 to cleave the RNA strand resolving the R-loops (Kang et al, 2021). In contrast, m 6 A in R-loop in some somatic cells promotes the formation of R-loops (Yang et al, 2019).…”

Consequences and Resolution of Transcription–Replication Conflicts

“…These include DHX9, DDX1, DDX3, DDX5, DDX19, DDX21, DDX23, DDX39B, DDX43, DDX47 and DDX56 [ 103 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 ]. TonEBP was also recently identified as an R-loop sensor and shown to recruit the RNA methyltransferase-like 3 (MTLL3) specifically to N6-methyladenosine (m6A) methylated R-loops [ 153 , 154 ]. This R-loop modification seems to play an important role in R-loop sensing and processing as it recruits RNase H for the degradation of the RNA [ 154 ].…”