| Summary: | Borealin is a novel fourth subunit of the vertebrate chromosomal passenger complex containing aurora-B kinase, INCENP, and surviving, which has essential regulatory roles at centromeres and the central spindle in mitosis. Co-immunoprecipitation experiments of endogenous proteins suggest that essential all of surviving, the majority of INCENP, and approximately half of aurora-B are complexed with borealin in mitotic cells. We also detected a sub-complex containing aurora-B and INCENP, but no borealin or surviving. Results from sucrose gradient sedimentation experiments suggest that there are high molecular weight complexes containing chromosomal passengers, which may contain nucleosomes. Binding experiments <i>in vitro</i> revealed a strong direct interaction of borealin with surviving, suggesting that it is the main binding partner of borealin in mitosis. Borealin also binds itself <i>n vitro,</i> and this interaction is detectable <i>in vivo</i>. We investigated the role of borealin within the complex and present evidence that, in contrast to INCENP and surviving, borealin is unlikely to be involved in the regulation of aurora-B kinase activity, but may be the subunit that targets the aurora-B and polo-like kinase 1, another essential mitotic kinase. Depletion of borealin by RNA interference delays mitotic progression and results in kinetochore-spindle mis-attachments and an increase in bipolar spindles associated with ectopic asters. The extra poles severely disrupt the partitioning of chromosomes in anaphase, revealing an unexpected role for the chromosomal passenger complex in the maintenance of mitotic spindle integrity. These studies have identified novel non-histone components of mitotic chromosomes, one of which, nuclear protein p30, is likely to play a role in centromeric chromatin structure.
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