New Roles of TonEBP in R-loop resolution

Abstract

R-loop is a novel three-stranded structure consisting of RNA-DNA hybrid and a displaced single-strand. R-loops are generated by transcription stalling derived from diverse inappropriate bioprocesses and play important roles in many cellular activities. The abnormal accumulation of R-loops can cause severe problems such as genomic instability and malignant diseases. Recently, it has been reported that R-loops are regulated or resolved through methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation. However, the detailed mechanism by which METTL3 is recruited to R-loop remains elusive. Here we demonstrated that tonicity-responsive enhancer binding protein (TonEBP) identifies R-loops using single-molecule imaging technique called as DNA curtain as well as biochemical assays. Using the DNA curtain, we found that TonEBP binds R-loops via both 3D collision and 1D diffusion along DNA. In addition, TonEBP moves along DNA not by hopping but by sliding without rotation around DNA helix. Biochemical assays showed that TonEBP preferentially binds to the displaced single-strand of R-loop, not to RNA-DNA hybrid structure. Moreover, we observed the colocalization of TonEBP and METTL3 at DNA damage. Depletion of TonEBP or METTL3 increases intracellular R-loop level and reduces cell survival. Our study revealed an R-loop resolution pathway by TonEBP and METTL3, providing new insights into R-loop resolution processes.