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ABSTRACT Evolutionary theory suggests that critical cellular structures should be subject to strong purifying selection as protein changes would result in inviability. However, how this evolutionary principle relates to multi-subunit complexes remains incompletely explored. For example, the macromolecular kinetochore complex, which mediates the faithful segregation of DNA during cell division, violates the expectation of purifying selection as subsets of kinetochore proteins exhibit rapid evolution despite its critical role. Here, we developed a multi-level approach to investigate the evolutionary dynamics of the kinetochore as a model for understanding how an essential multi-protein structure can experience high rates of diversifying selection while maintaining function. Our comprehensive approach analyzed 57 kinetochore genes for signatures of purifying and diversifying selection across 70 mammalian species. Intraspecies comparisons of kinetochore gene evolution showed that members of the order Afrotheria experience higher rates of diversifying selection than other mammalian orders. Among individual loci, genes that serve regulatory functions, such as the mitotic checkpoint genes, are conserved under strong purifying selection. In contrast, the proteins that serve as the structural base of the kinetochore, including the inner and outer kinetochore, evolve rapidly across species. We also demonstrated that diversifying selection is targeted to protein regions that lack clear structural predictions. Finally, we identified sites that exhibit corresponding trends in evolution across different genes, potentially providing evidence of compensatory evolution in this complex. Together, our study of the kinetochore reveals a potential avenue by which selection can alter the genes that comprise an essential cellular complex without compromising its function. 

Summary The cohesin complex is critical for genome regulation, relying on specialized co-factors to mediate its diverse functional activities. Here, by analyzing patterns of similar gene requirements across cell lines, we identify PRR12 as a regulator of cohesin and genome integrity. We show that PRR12 interacts with cohesin and PRR12 loss results in a reduction of nuclear-localized cohesin and an accumulation of DNA lesions. We find that different cell lines across human and mouse exhibit significant variation in their sensitivity to PRR12 loss. Unlike the modest phenotypes observed in human cell lines, PRR12 depletion in mouse cells results in substantial genome instability. Despite a modest requirement in human cell lines, mutations in PRR12 lead to severe developmental defects in human patients, suggesting context-specific roles in cohesin regulation. By harnessing comparative studies across species and cell lines, our work reveals critical insights into how cohesin is regulated across diverse cellular contexts. 

Similar to other core biological processes, the vast majority of cell division components are essential for viability across human cell lines. However, recent genome-wide screens have identified a number of proteins that exhibit cell line–specific essentiality. Defining the behaviors of these proteins is critical to our understanding of complex biological processes. Here, we harness differential essentiality to reveal the contributions of the four-subunit centromere-localized CENP-O complex, whose precise function has been difficult to define. Our results support a model in which the CENP-O complex and BUB1 act in parallel pathways to recruit a threshold level of PLK1 to mitotic kinetochores, ensuring accurate chromosome segregation. We demonstrate that targeted changes to either pathway sensitizes cells to the loss of the other component, resulting in cell-state dependent requirements. This approach also highlights the advantage of comparing phenotypes across diverse cell lines to define critical functional contributions and behaviors that could be exploited for the targeted treatment of disease.