Biology subjects

Nishikura, K.

Publications and source records attributed to Nishikura, K..

2 recordsLinked to original sources

bioRxiv2025

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

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.

molecular biology↗

bioRxiv2025

LANA-Dependent Transcription-Replication Conflicts and R-Loops at the Terminal Repeats (TR) Correlate with KSHV Episome Maintenance

Transcription-replication conflicts frequently occur at repetitive DNA elements involved in genome maintenance functions. The KSHV terminal repeats (TR) function as the viral episome maintenance element when bound by the viral encoded nuclear antigen LANA. Here, we show that transcription-replication conflicts occur at or near LANA binding sites in the TR. We show by proximity ligation assay (PLA) that PCNA and RNAPII colocalize with LANA-nuclear bodies (LANA-NBs). Using DNA-RNA-IP (DRIP) assays with S9.6 antibody, we demonstrate that R-loops form at the TR. We find that these R-loops are also associated with histone H3pS10 a marker for R-loops associated with transcription-replication conflicts. Inhibitors of RNA polymerase eliminated LANA binding to the TR, along with the loss of R-loops and activation associated histone modifications, and the accumulation of heterochromatic marks. We show that LANA can induce all of these features on a plasmid containing 8, but not 2 copies of the TR, correlating strongly with episome maintenance function. Taken together, our study indicates that LANA induces histone modifications associated with RNA and DNA polymerase activity and the formation of R-loops that correlate with episome maintenance function. These findings provide new insights into mechanisms of KSHV episome maintenance during latency and more generally for genome maintenance of repetitive DNA. ImportanceKSHV latent infection is responsible for Kaposis Sarcoma (KS) and Pleural Effusion Lymphoma (PEL). KSHV latency and persistence depends on LANA binding to the terminal repeats (TR). We show that LANA binding promotes the formation of R-loops associated with transcription-replication conflicts and histone H3pS10 at the KSHV terminal repeats. These epigenetic features depend on active RNA polymerase at the TR and correlate strongly with KSHV episome maintenance function. The findings suggest a novel mechanism of chromatin structural maintenance dependent on LANA binding at the TR during KSHV latency.

molecular biology↗