Abstract
Ccr4-Not protein complexes are evolutionarily conserved and have important functions in mRNA regulatory processes. Strict regulation of mRNA’s is important for cells since they form the central connection between the genetic information in the nucleus and active protein production in the cytoplasm. Ccr4-Not complexes are involved in mRNA synthesis and
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degradation, histone methylation and protein degradation via the ubiquitin-proteasome pathway. The emerging picture for the Ccr4-Not complex is that of a regulatory platform with distinct enzymatic activities that controls important cellular processes such as cell proliferation, adaptations to environmental changes and normal embryogenesis. The aim of the studies described in this thesis is to gain more insight in the functions and regulation of the multi-subunit Ccr4-Not complex. This was performed by studying the composition and localization of human Ccr4-Not complexes, and the phosphorylation of the Ccr4-Not complex in budding yeast. The composition of the Ccr4-Not complex is well-established in yeast, but unknown in human cells with their multiple deadenylases. The composition and protein interactors of human Ccr4-Not complexes have been identified in stable cell lines with near endogenous expressed Ccr4-Not subunits using a combination of biochemical and in-depth proteomic approaches. At least four distinct Ccr4-Not complexes differing in their deadenylase subunits are identified in human cells and CNOT4 resides in a separate protein complex. Involvement of the Ccr4-Not complexes in splicing, transport and localization of RNA molecules is suggested by their associated protein interactors. Furthermore, spatial organisation of human GFP-fused Ccr4-Not subunits at endogenous levels has been visualized in stable cell lines by (real-time) fluorescence microscopy. The CNOT proteins localize predominantly to the cytoplasm, with the exception of a cell cycle-dependent nuclear-cytoplasmic distribution of CNOT7 proteins. Moreover, the spatial regulation of Ccr4-Not proteins was explored in a pilot study using leptomycin B. More CNOT7, CNOT8 and CNOT9 proteins were detected in the nucleus upon inhibition of NES-mediated nuclear protein export by this drug. Interestingly, these three Ccr4-Not subunits harbour a predicted NES. Finally, since Not4p and Bur2p of the Bur1/2p kinase complex genetically interact, Not4p phosphorylation in relation to Bur2p was investigated in yeast using a combination of molecular biology, biochemistry, mass spectrometry, phenotypical assays and genetics. Not4p was confirmed as a phospho-protein whose phosphorylation sites are targets of kinases other than Bur1p, indicating that the Bur1/2p kinase complex is not directly involved in Not4p phosphorylation. Both Not4p and Bur2p are required for global H3K4me3 marks, but disruption of all known Not4p phosphorylation sites maintains normal H3K4me3 levels. Phenotypical assays showed that the five phospho-sites on Not4p are important for resistance to cellular stresses in molecular pathways parallel to BUR2. In conclusion, eukaryotic Ccr4-Not protein complexes may be a general mRNA metabolism regulator. The relative contributions of variable Ccr4-Not (sub)complexes in distinct gene expression processes will be an important subject for future studies
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