JM_ETHZ_IEF_2009

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

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 Obiettivo del progetto (Objective)

'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 However, unpublished data from the Peter laboratory suggest that CRL4s may also be important for progression through mitosis.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.'

Introduzione (Teaser)

Mitotic cell division is a critical stage in the cell cycle process with any errors having potentially fatal consequences, such as cancer. EU-funded research investigated the role of a subfamily of enzymes in maintaining the fidelity of genetic material down the generations .

Descrizione progetto (Article)

Cullin4A-RING E3 ubiquitin ligases (CRL4s) are protein complexes that play an important role during DNA replication, chromosome segregation and controlled protein breakdown to ensure genome integrity. During mitosis, post-translational modifications such as ubiquitination (addition of ubiquitin molecule fragments) play a key role in protein regulation and localisation.

The project 'Function and regulation of CRL4s-based ubiquitin-dependent processes for mitotic progression' (JM_ETHZ_IEF_2009) was initiated to study how CRL4s regulate mitotic processes in our cells. In particular they looked at enzymes involved in the ubiquitination process.

Researchers carried out automated high-content live cell imaging with RNAi-based screen using advanced microscopy. This permitted the monitoring of individual cells throughout the cell cycle and helped in the identification of CRL4 substrate adaptors. Two promising candidates were subjected to comprehensive functional and biochemical analysis.

Study results revealed that one of these acted on motor proteins and helped regulate chromosome congression during mitosis. The other candidate ensured accurate chromosome alignment during kinetochore formation and establishment in mitosis. The kinetochore is a protein complex that acts as attachment sites for spindle microtubules during mitosis to properly align chromosomes.

Project activities have demonstrated that CRL4s play an important role in regulating mitosis and ensuring error-free progression through ubiquitination of key proteins. Future research activities could focus on pinpointing the physiological substrates of these adaptors. This work would be of huge significance in diagnosing and developing gene-based therapies for diseases like cancer and genetic disorders.