Ultra-structural and dynamic analyses of the sub-cellular organisation of messenger RNA biology

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Messenger RNA is crucial to cellular gene expression because it interprets genetic information encoded by DNA. The mechanisms involved include two major events of mRNA production and use. Firstly, splicing is particularly important because it creates mature mRNA molecules and allows flexibility of the genome by creating different splicing isoforms. Secondly, sub-cellular transport of mRNA has been shown to be vital to embryonic development in species such as Drosophila, as well as also regulating pluripotency of mouse embryonic stem cells.Henceforth, the physical organisation of mRNA splicing within the nucleus of a cell has not been fully understood. Hence, this project aims to gain an insight into some of the structures involved in alternative splicing and mRNA trafficking. Cells from the eye lens will be used because they present an effective model of cellular development. This is because, as cells in the lens persist throughout the life span of an organism, the model provides an entire developmental history to examine. Furthermore, structures within the nucleus specific to RNA processing, show distinct patterns of alteration during lens development. MBNL1 and CELF1 proteins are alternative splicing factors that have antagonistic roles in controlling the developmental switch between foetal and adult isoforms. Furthermore, MBNL1 has been shown to co-localise with mRNA in the cytoplasm and regulate its localisation. Therefore, these structures will be mostly the focus of this investigation. Previously, the protein structures have been tagged with GFP and the recently innovative molecule: mini Singlet Oxygen Generator (miniSOG). This has allowed one to observe the localisation of these structures within the nucleus and cytoplasm. Now, the aim is to examine their sub-cellular dynamics in lens epithelial cells, intact eye lenses and differentiated muscle cell lines. This will allow one to analyse the antagonistic effects of MBNL1 and CELF1 within the splice-site and mRNA localisation in the cytoplasm. Techniques to be used will include those in Microscopy (particularly Fluorescence, Electron, Live Cell Time Lapse, Photobleaching and FRET), Molecular Biology, Biochemistry and Mass spectrometry.