B Since localization of Aurora B is essential for its function, and Aurora B levels were not regulated by UBASH3B, we next analyzed localization of Aurora B in the presence and absence of UBASH3B. Downregulation of MedChemExpress Scutellarein UBASH3B by siRNA led to the redistribution of the centromeric Aurora B to the chromosome arms in prometaphase cells and the total chromosome area occupancy by Aurora B was increased. The same localization defects of Aurora B were also observed in UBASH3Bdepleted cells arrested in prometaphase by the Eg5 inhibitor STLC. In addition, downregulation of UBASH3B in prometaphase-arrested cells also PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19812666 led to spreading of other components of the CPC including Survivin and INCENP to chromosomal arms, consistent with their mutual co-regulation with Aurora B. These data suggest that UBASH3B regulates Aurora B localization in early mitotic stages. How can spindle associated UBASH3B control the chromosomal distribution of Aurora B Interestingly, spindle microtubules had been shown to regulate centromeric focusing of Aurora B, which also requires the Aurora Bdependent positive feedback loops. Thus, UBASH3B may provide a molecular link between the centromeric and the microtubule-associated fractions of Aurora B. To test this assumption, we analyzed Aurora B localization in the later mitotic stages. Strikingly, downregulation of UBASH3B reduced localization of Aurora B to the midzone microtubules; the bulk of Aurora B remained associated with the arms of segregating chromosomes throughout anaphase. Next, we tested if the Aurora B localization defects were specific to downregulation of UBASH3B. Importantly, re- Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Dev Cell. Author manuscript; available in PMC 2017 April 21. Krupina et al. Page 6 expression of FLAG-tagged UBASH3B at nearly endogenous levels reversed the observed localization defects in prometaphase. The ubiquitin binding of UBASH3B plays a crucial role in this process as the form of UBASH3B mutated in UBA and eUBA domains did not rescue the localization defects of Aurora B. The delay from prophase to anaphase in UBASH3B downregulated cells was also reversed by re-expression of the wild-type form in UBASH3B-depleted cells. The lagging anaphase chromosomes observed in UBASH3B siRNA cells were markedly decreased in cells expressing wild type but not the mutant UBASH3B. Likewise, 
the multilobed nuclei phenotype observed upon downregulation of UBASH3B was dependent on ubiquitin binding by UBASH3B. Collectively, UBASH3B specifically regulates localization of Aurora B and mitotic progression in ubiquitindepentent manner. Interestingly, the Aurora B localization defects are very similar to those observed upon inactivation of CUL3 E3 ubiquitin ligases. Next, we dissected the roles of the chromatin and microtubule pathways involved in the centromeric focusing of Aurora B. UBASH3B siRNA treatment led to the chromosomal spreading of active centromeric Aurora B kinase visualized by an antibody to the phosphorylated catalytic domain of Aurora kinases , suggesting that Aurora B-mediated positive feedback loop signaling may also be dependent on UBASH3B. Specifically, Aurora B is known to phosphorylate Haspin kinase to promote generation of Threonine 3-phosphorylated Histone H3 thereby enhancing its own recruitment to the centromeres. Indeed, depletion of UBASH3B markedly reduced the levels of P-T3-H3 around the centromeric regions but did not affect the Serine 10-phosphory