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.
Highlights
R-loops are three-stranded nucleic acid structures consisting of a DNA-RNA hybrid and a single-stranded DNA
Combining tandem affinity purification and mass spectrometry, we identified proteins that interact with the N terminus of tonicity-responsive enhancer binding protein (TonEBP) (TonEBP Yc1 domain: Yc1), which encompasses the entire Rel homology domain (RHD) of TonEBP
Because the RHD of TonEBP has conserved amino acids sequences, we produced two TonEBP mutant proteins carrying point mutations (3M and 5M) at the highly conserved and charged amino acids (Figure 1H). 3M is a mutant of Yc1 in which R, E and R are all replaced by A shown in red and 5M is another mutant where K, R and the three Ks are all exchanged with A shown in green
Summary
R-loops are three-stranded nucleic acid structures consisting of a DNA-RNA hybrid and a single-stranded (ss) DNA. R-loops have pleiotropic functions essential for eukaryotic physiology; they are important for chromosome segregation in mitosis, immunoglobulin class switching, DNA replication and repair, and transcription [1,2,3]. These structures have biological relevance in regulating gene expression and specialized rearrangement events [4,5]. R-loop accumulation is associated with a variety of diseases involving genomic instability, including myelodysplastic syndromes, neurodegenerative diseases, and cancers such as Ewing’s sarcoma [5,6,7]. Many factors have been identified for R-loop generation and resolution [8,9,10,11], it is
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