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Function and regulation of CRL4s-based ubiquitin-dependent processes for mitotic progression

Final Report Summary - JM_ETHZ_IEF_2009 (Function and regulation of CRL4s-based ubiquitin-dependent processes for mitotic progression.)

Mitosis corresponds to a brief but essential part of cell cycle. During this highly dynamic period, replicated DNA is equally segregated between the two daughter cells and cell morphology is dramatically changing. These changes do not only take place at the cellular levels, but also include changes in expression levels, destruction, and modification of many various proteins. The spatial and temporal regulation of mitosis is the focus of intensive research, as errors in these regulatory systems correlate with many human diseases including cancer. Therefore, it is important to understand the molecular mechanisms that govern faithful DNA replication and chromosome segregation.

During mitosis, proper chromosome alignment at the metaphase plate requires that spindle microtubules correctly attach to specific chromatin structure -- the kinetochores. Segregation in case of incorrect attachment could lead to deleterious effects on cells, such as chromosome missegregation, aneuploidy and DNA breakages. Microtubule-kinetochore attachment is a highly dynamic process that depends on self-organization properties coupled to mechanisms that coordinate microtubules, kinetochores and motor proteins. Post-translational modifications are major contributors to protein regulation in space and time. In particular, ubiquitination -- the addition of ubiquitin moieties to specific protein substrates -- could directly influence protein lifetime, as well as its localization or its interaction with other proteins. Ubiquitination is a stepwise mechanism that involves the sequential action of dedicated enzymes, ultimately recognizing and transferring ubiquitin to the target protein. Among the hundreds of enzymes known so far able to transfer ubiquitin to target proteins, cullin-based E3 ligases emerged as major components accounting approximately for one third of all ubiquitin ligase enzymes, thereby regulating a wide range of cellular functions throughout the cell cycle.

In this project, we investigated how a subfamily of cullin-containing enzymes could affect mitotic processes in human cells. We performed a high-content live cell imaging RNAi-based screen with putative substrate receptors functioning in cullin-based E3-ligases. Specifically, we used automated high content live cell microscopy to monitor the progression of individual cells through the cell cycle. From this screen, we obtained a set of promising candidates that disrupt mitosis upon depletion of the protein by RNAi. We expect that investigating the molecular function of these putative substrate adaptors may help to better understand the mechanisms that ensure error-free progression through mitosis.

In the frame of this project, we performed a detailed functional and biochemical analysis of two candidates identified in the screen. One appears to act on motor proteins impacting chromosome congression, whereas the second one is required for proper centromeric DNA formation, essential for kinetochore establishment and thus microtubules attachment to mitotic chromosomes. The outcome of this project thus suggests that ubiquitination of key proteins regulates these critical processes, and future experiments now focus on the identification of the physiological substrates of these adaptors.