Mitosis relies heavily on ubiquitin-dependent degradation of specific protein substrates, mainly by the APC/C and CUL3-RING E3 ligases (CRL3s). In contrast, CUL4-RING E3 ligases (CRL4s) have previously been associated with silencing of gene expression, DNA replication and repair. However, unpublished data from the Peter laboratory suggest that CRL4s may also be important for progression through mitosis. In this grant proposal, I thus present two complementary approaches to investigate the role of CRL4s for progression through mitosis in human cells. First, I will complete an RNAi screen to identify specific CRL4 substrate adaptors and regulators important for correct mitotic progression. Second, I will functionally and biochemically characterize the most promising candidates, which may regulate kinetochore function and faithful chromosome alignment. Preliminary results from a pilot screen performed in our lab, as well as the on-going screen, already indicate several promising candidates required for faithful progression through mitosis. By combining an unbiased large-scale with a candidate approach, I expect to unravel novel functions of CRL4s in mitosis, and identify specific roles for CRL4 substrate adaptors in spindle/microtubules dynamics, kinetochore function, and chromosome attachment. I believe that the proposed experiments cover an important area of fundamental biochemistry and cell biology and will provide important new insights into our understanding of mitosis.
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