Dual roles of N⁶-methyladenosine in R-loop regulation of gene transcription and genome stability

N⁶-methyladenosine (m⁶A) in RNA within R-loops plays pivotal roles in transcription regulation and genome stability. However, the precise impacts and distinct mechanisms of m⁶A on both regulatory and aberrant R-loops remain poorly understood. Here, we reveal that METTL3, the nuclear m⁶A writer, ensures genome integrity by differentially modulating R-loops in a position- and length-dependent manner. In mouse embryonic stem cells (mESCs), Mettl3 depletion results in impaired cell proliferation and increased cell death due to excessive DNA damage. Notably, Mettl3 knockout reduces the overall abundance of R-loops, with a decrease in broad R-loops and an increase in sharp R-loops. R-loops are diminished near transcription end sites (TESs), leading to transcriptional readthrough of genes with m⁶A-modified transcripts and potentially contributing to genome instability. Conversely, increased sharp R-loops located in the antisense orientation relative to gene transcription are associated with DNA damage hotspots. These findings unveil a dual regulatory mechanism in which METTL3-m⁶A orchestrates transcription fidelity and genome stability through distinct R-loop-dependent manners.