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

信使RNA对细胞基因表达至关重要，因为它解释由DNA编码的遗传信息。所涉及的机制包括mRNA的产生和使用两个主要事件。首先，剪接是特别重要的，因为它创造了成熟的mRNA分子，并通过创造不同的剪接异构体来允许基因组的灵活性。其次，mRNA的亚细胞转运已被证明对果蝇等物种的胚胎发育至关重要，同时也调节小鼠胚胎干细胞的多能性。此后，细胞核内mRNA剪接的物理组织尚未完全了解。因此，本项目旨在深入了解参与选择性剪接和mRNA运输的一些结构。将使用眼晶状体细胞，因为它们是细胞发育的有效模型。这是因为，由于晶状体中的细胞在生物体的整个生命周期中持续存在，该模型提供了一个完整的发育历史来研究。此外，在晶状体发育过程中，细胞核内特定于RNA加工的结构显示出明显的改变模式。MBNL1和CELF1蛋白是可选择的剪接因子，在控制胎儿和成人亚型之间的发育转换中具有拮抗作用。此外，MBNL1已被证明与细胞质中的mRNA共定位并调节其定位。因此，这些结构将主要是本研究的重点。此前，这些蛋白质结构已经被绿色荧光蛋白和最近的创新分子：迷你单线态氧气发生器（miniSOG）标记。这使得人们可以观察到这些结构在细胞核和细胞质中的定位。现在，我们的目标是研究它们在晶状体上皮细胞、完整的眼晶状体和分化的肌肉细胞系中的亚细胞动力学。这将允许人们分析MBNL1和CELF1在剪接位点和细胞质中mRNA定位中的拮抗作用。将使用的技术将包括显微镜技术（特别是荧光、电子、活细胞延时、光漂白和FRET）、分子生物学、生物化学和质谱。

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.