Spatial Mapping and Interactome Profiling of m ⁶ A-Modified R-Loops via Chemically Inducible Split-APEX2 Proximity Labeling

m⁶A-Modified R-loops (m⁶A-R-loops) play crucial roles in epigenetic regulation and genome stability, yet resolving their spatial distribution and protein interactomes in live cells remains challenging. To address this, we developed m⁶A-R-loop proximity labeling (m⁶A-RLPL), a chemically inducible split-APEX2 proximity labeling technology integrating dual-target recognition using the RNA-DNA hybrid binding domain of RNase H1 for R-loop targeting and m⁶A reader protein's YTH domain for m⁶A recognition, coupled with an abscisic acid (ABA)-inducible dimerization system for signal amplification. This technology revealed host m⁶A-R-loops enriched with nucleoli under normal conditions. When applied to herpes simplex virus (HSV) infection, it further demonstrated viral m⁶A-R-loops undergoing dramatic accumulation within phase-separated granules in replication compartments during late-stage infection. Proximity proteomics identified ZC3H4 and CCDC124 as essential regulators maintaining these structures, which serve as transcription sites for HSV late genes, with disruption selectively impairing viral transcription. m⁶A-RLPL establishes a generalizable approach for spatially resolved profiling of m⁶A-R-loop interactomes and organizational dynamics in living systems.