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Spatial Mapping and Interactome Profiling of m6A-Modified R-Loops via Chemically Inducible Split-APEX2 Proximity Labeling.

Meng-Die Zhu·Hua-Qian Yin·Zhong-Da He·Yao Cheng·Qing-Qing Ye·Zhi-Gang Wang·Dai-Wen Pang·Shu-Lin Liu
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Abstract

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.

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HumansR-Loop StructuresAdenosineRibonuclease HAnimalsJournal ArticleResearch Support, Non-U.S. Gov't

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Meng-Die Zhu, Hua-Qian Yin, Zhong-Da He, Yao Cheng, Qing-Qing Ye, Zhi-Gang Wang, Dai-Wen Pang, Shu-Lin Liu. 2025-12-03. Spatial Mapping and Interactome Profiling of m6A-Modified R-Loops via Chemically Inducible Split-APEX2 Proximity Labeling.. https://doi.org/10.1021/acs.analchem.5c05501

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