Speaker
Description
The correct association of kinetochores with microtubules connecting opposite spindle poles is essential for accurately segregating sister chromatids during mitosis. To gain mechanistic insights into this dynamic process, it is important to understand the roles of the various complexes that form the multi-layered kinetochore. The CENP-OPQUR is a sub-complex of the kinetochore. Its depletion affects chromosome congression and leads to mitotic delay. However, traditional methods of depleting CENP-OPQUR in cells, such as RNA interference (RNAi), typically take several days, potentially undermining the relevance of these observations. To tackle this challenge, we established a robust acute degradation system through a CRISPR/Cas9-mediated knock-in approach to bi-allelically tag CENP-U with an FKBPF36V tag, enabling rapid degradation of the endogenous protein upon treatment with nanomolar concentrations of dTAGV-1, allowing us to visualize the immediate consequences of CENP-OPQUR degradation. The Cohesin complex is vital for maintaining sister chromatid cohesion at the centromere during mitosis and has been suggested to have a kinetochore-specific pool. In this study, we identified CENP-OPQUR as the primary kinetochore receptor for the Cohesin complex and successfully reconstituted this interaction in vitro using purified protein complexes. Through site-directed mutagenesis, we conclusively demonstrated that the canonical FxF binding motif of the CENP-U subunit directly interacts with the composite binding site formed by the SA2 and SCC1 subunits of the Cohesin complex. Moreover, we established that CENP-OPQUR is the primary receptor of the Cohesin complex at the inner kinetochore. This newly identified interaction opens up avenues for a deeper understanding of the functions of CENP-OPQUR complex in maintaining sister chromatid cohesion and shaping the architecture of the centromere through Cohesin loading at the kinetochore, thereby providing insights into the regulatory mechanisms of mitotic progression by the kinetochore.
