Polo kinase recruitment via the constitutive centromere-associated network at the kinetochore elevates centromeric RNA.

Author summary During cell division, replicated chromosomes must be equally divided between the two daughter cells. This is achieved in part by a multi-protein structure called the kinetochore. Errors during chromosome segregation can lead to inheritance of abnormal chromosome number. This situation is referred to as aneuploidy-a hallmark of cancer cells. Kinetochore function is tightly regulated by recruitment of regulatory proteins under certain conditions and at specific times during the cell cycle. Kinetochore regulators are often mis-regulated in many types of cancers. The major cell-cycle regulator, Polo-like kinase (Plk1), is one such regulator. Plk1 is overexpressed in many cancers and is thus considered an important target in cancer therapeutics. To understand its function in kinetochore regulation, we manipulated the Plk1 homolog in budding yeast, Cdc5, in both space and time. Our data suggest that Cdc5 has multiple different functions at the yeast kinetochore in agreement with studies in human cells. Furthermore, we show that constitutive Cdc5 localization at the inner kinetochore can disrupt the function of the whole kinetochore, perturb mitotic progression and increase the levels of centromeric RNA. These results have implications for human disease, since mis-regulation of centromeric transcription has been observed in stressed, aging and cancerous cells. The kinetochore, a multi-protein complex assembled on centromeres, is essential to segregate chromosomes during cell division. Deficiencies in kinetochore function can lead to chromosomal instability and aneuploidy-a hallmark of cancer cells. Kinetochore function is controlled by recruitment of regulatory proteins, many of which have been documented, however their function often remains uncharacterized and many are yet to be identified. To identify candidates of kinetochore regulation we used a proteome-wide protein association strategy in budding yeast and detected many proteins that are involved in post-translational modifications such as kinases, phosphatases and histone modifiers. We focused on the Polo-like kinase, Cdc5, and interrogated which cellular components were sensitive to constitutive Cdc5 localization. The kinetochore is particularly sensitive to constitutive Cdc5 kinase activity. Targeting Cdc5 to different kinetochore subcomplexes produced diverse phenotypes, consistent with multiple distinct functions at the kinetochore. We show that targeting Cdc5 to the inner kinetochore, the constitutive centromere-associated network (CCAN), increases the levels of centromeric RNA via anSPT4dependent mechanism.