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SUMMARY Mesendodermal cells are key intermediate progenitors that form the early primitive streak (PrS) and give rise to mesoderm and endoderm in the gastrulating embryo. We have identified an interaction between the CCR4-NOT complex member CNOT3 and the cell cycle kinase Aurora B that regulates MAPK/ERK signalling during mesendodermal differentiation. Aurora B phosphorylates CNOT3 at two sites that are located close to a nuclear localization signal and promotes localisation of CNOT3 to the nucleus in mouse ES cells (ESCs). Mutation of these sites in ESCs gives reduced numbers of embryoid bodies that are largely composed of ectoderm and interferes with differentiation of ESCs into mesendoderm in response to FGF2, BMP4, Wnt3 and Activin. The double mutation affects interaction of CNOT3 with Aurora B and ERK and reduces phosphorylation of ERK in response to FGF2. Our results identify a signalling axis involving CNOT3 that regulates a key pathway during embryogenesis.

Gene expression is a process that is tightly regulated by many factors. Different genes are transcribed not only in a cell specific manner but are also differentially expressed at different stages of the cell cycle. Cnot3 is part of the CCR4-NOT deadenylation complex, which is involved in the turnover of mRNAs in the cytoplasm and has also been shown to have roles in regulating transcription and cell proliferation and in maintaining ES cell pluripotency. Previous work demonstrated that Cnot3 interacts directly with Aurora B kinase and is phosphorylated by Aurora B in an in vitro assay. Aurora B and Cnot3 co-localise at active gene promoters in resting B cells. Since Aurora B is a cell cycle kinase, I have developed a cell synchronization system to analyse the role of the Cnot3-Aurora B interaction at different stages of the cell cycle in primary B cells. Using this system, I have demonstrated that the interaction between Cnot3 and Aurora B varies during cell cycle progression. In vitro analysis showed that the interaction occurs through the NOT box domain of Cnot3. Mass spectrometry analysis of Cnot3 interactors, performed on nuclear extracts from B cells in the early G1 and G2 phases of the cell cycle, identified interactions with many factors that are known to have roles in transcription regulation and RNA processing. Interaction of Cnot3 with Histone H1 was confirmed using a peptide binding assay, suggesting a potential role in chromatin organization. Cnot3 was also shown to interact with Xrn2, a 5’-3’ exoribonuclease that is involved in RNA turnover and termination of transcription. ChIP analysis demonstrated promoter binding of Cnot3 at a number of cell cycle stages. Cnot3 shows cell cycle dependent binding to promoters of a wide range of active genes, including promoters that are not directly involved in cell cycle regulation. Genome wide analysis using ChIP sequencing revealed changes in the binding profiles of Cnot3 at promoters and enhancers during cell cycle progression. A Cnot3 conditional knock out mouse has been generated, which will be used to test the functional importance of these observations. Open Access