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Lee, Ja Yil
Biochemistry and Molecular Biophysics Lab.
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TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution

Cheon, Na YoungKang, Hyun JeMyung, KyungjaeKwon, Hyug MooLee, Ja Yil
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한국물리학회 2020 가을학술대회
R-loop is a hybrid structure consisting of RNA-DNA hybrid and displaced single-stranded DNA. R-loops are induced by inappropriate transcription, transcription-replication collision and DNA damage. Normally R-loops play important roles in many cellular activities, but the abnormal accumulation of R-loops also can cause serious problems such as genomic instability or cancer. To regulate R-loops, methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation resolve it. However, the detailed mechanism by which R-loop is recognized and METTL3 is recruited to R-loop remains unclear. Here, using single-molecule imaging technique DNA curtain and other biochemical assays, we find that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops. In DNA curtain assays, we observed that TonEBP binds R-loops via both 1D diffusion and 3D collision. Furthermore, we demonstrate that TonEBP moves along DNA by sliding mechanism without helical rotation from salt-independent diffusion coefficients. Furthermore, we reveal that TonEBP binds the displaced ssDNA strand of R-loop, not RNA-DNA hybrid structure using electrophoretic mobility shift assay. Next, we examine the interaction between TonEBP and METTL3 that catalyzes methylation of A6 in RNA. We observe the colocalization of TonEBP and METTL3 at DNA damage site. Depletion of TonEBP and METTL3 increases R-loops and reduced cell survival. Our study reveals an R-loop resolution pathway by TonEBP and METTL3 and provide new insights into R-loop resolution processes.


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