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bioRxiv · 10.1101/2025.05.28.656747

Mitotic phosphorylation of ADAR1 regulates its centromeric localization and is required for faithful mitotic progression

Yang, Y.·Kubota, M.·Hasegawa, K.·Zeng, C.·Nishikura, K.·Hamada, M.·Sakurai, M.
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Abstract

Adenosine Deaminase Acting on RNA 1 p110 (ADAR1p110), the constitutively expressed nuclear isoform of the RNA-editing enzyme ADAR1, plays well-established roles in adenosine-to-inosine RNA editing, but its functions during mitosis remain poorly defined. Here, we identify ADAR1p110 as a chromatin-associated factor essential for mitotic chromosome segregation and cell viability. Depletion of ADAR1 caused metaphase arrest, DNA damage accumulation, and apoptosis. Co-immunoprecipitation experiments revealed that ADAR1p110 physically interacts with Structural Maintenance of Chromosome 3 (SMC3), a core component of the cohesin complex. Genome-wide DNA immunoprecipitation sequencing (DIP-seq) showed that ADAR1p110 selectively binds centromeric -satellite DNA during mitosis, a finding validated by DNA immunoprecipitation quantitative polymerase chain reaction (DIP-qPCR). Complementary DNA-RNA immunoprecipitation sequencing (DRIP-seq) revealed that RNA:DNA hybrids (R-loops) are enriched in centromeric regions during mitosis and are further augmented by ADAR1 overexpression. We identified serine 614 (S614) as a key mitosis-specific phosphorylation site on ADAR1p110, and demonstrated that this post-translational modification is essential for its chromatin recruitment, stability, and mitotic function. Rescue experiments using phospho-mimetic mutants (S614D and 3xD) successfully restored mitotic progression in ADAR1-deficient cells, whereas non-phosphorylatable variants failed to do so. These results reveal that ADAR1p110 is phosphorylated in a cell cycle-dependent manner and functions at the intersection of post-transcriptional and post-translational regulation. By coordinating R-loop recognition at centromeres with chromosome cohesion, ADAR1p110 safeguards genome integrity during mitosis. Our findings uncover a previously uncharacterized mechanism through which a canonical RNA-editing enzyme contributes to chromosomal dynamics independent of its deaminase activity.

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Yang, Y., Kubota, M., Hasegawa, K., Zeng, C., Nishikura, K., Hamada, M., Sakurai, M.. 2025-05-30. Mitotic phosphorylation of ADAR1 regulates its centromeric localization and is required for faithful mitotic progression. https://doi.org/10.1101/2025.05.28.656747

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