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Dysregulation of translation has a direct effect on growth control in mammalian cells. The initiation step of translation is an important point of control of gene expression and a rate-limiting step for protein synthesis. Initiation requires initiation factors (eIFs) that are important for the activation of both mRNA and the recruitment of ribosomal subunits. Previously, post-translational modifications (PTMs), such as phosphorylation, have been shown to be an important part in fine-tuning translation. SUMOylation is another PTM that affects a number of fundamental cellular processes, including the response to DNA damage, metabolic regulation and protein trafficking. Many eIFs have been identified in proteomic screens as SUMOylated targets, but to date most of these modifications have not been confirmed. Preliminary work from the Watts lab indicated that some eIFs co-purified with the S. pombe SUMO protease Ulp2. The aim of my project was to determine whether components of the eIF4F complex are SUMOylated and to initiate studies to investigate the role of this modification. The first results chapter investigates eIF4G, eIF3h and Sla1 (the La protein homologue) and demonstrates that eIF4G and Sla1, but not eIF3h, are SUMOylated in S. pombe. These experiments were then extended to mammalian cells. The effects of stress conditions on protein synthesis and SUMOylation in a range of cell lines were first analysed. SUMO localisation was altered in response to sodium arsenite (AR) and ionising radiation (IR). In most cell types tested, for example, after IR treatment, SUMO1 went to nuclear foci in HeLa cells, but was more abundant in the cytoplasm following exposure to AR. Next, in vivo and in vitro SUMOylation assays were used to demonstrate that mammalian eIF4G and eIF4A are both SUMOylated. Mass spectrometric analysis identified the SUMOylation sites in eIF4G, as K1386 and K1588. Those of eIF4AI and eIF4AII are K225 and K226, respectively. Mutated eIF4AII was introduced into cells to investigate the role of SUMOylation of this factor. Colocalisation of eIF4A/eIF4G and SUMO1 shows that, in AR-treated cells, SUMO1 colocalises with eIF4A and eIF4G in the cytoplasmic stress granules, especially at their edges. In contrast, in IR-treated cells, the colocalisation of eIF4G/eIF4A with SUMO1 is much more in the nucleus, compared to that in untreated cells, suggesting that eIF4G/eIF4A and SUMO1 may have a cellular role in some aspects in response to AR and IR. |
