Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
Methods. 2014 Feb;65(3):359-66. doi: 10.1016/j.ymeth.2013.08.010. Epub 2013 Aug 19.
The ability to observe protein dynamics in living cells is critical for the mechanistic understanding of highly flexible biological processes such as pre-mRNA splicing by the spliceosome. Splicing relies on intricate RNA and protein networks that are repeatedly rearranged during spliceosome assembly. Here we describe a method based on fluorescence microscopy that has been used by our and other laboratories to study interaction of spliceosomal proteins with nascent pre-mRNA in living cells. The method involves co-expressing in mammalian cells the target pre-mRNA labeled with one color, and the spliceosomal protein tagged with another color. The diffusion coefficient of the protein as well as its association and dissociation rates with the pre-mRNA are estimated by fluorescence recovery after photobleaching (FRAP) or photoactivation.
在活细胞中观察蛋白质动态的能力对于理解剪接体进行的高度灵活的生物过程（如前体 mRNA 的剪接）的机制至关重要。剪接依赖于复杂的 RNA 和蛋白质网络，这些网络在剪接体组装过程中反复重排。在这里，我们描述了一种基于荧光显微镜的方法，我们和其他实验室已经使用该方法来研究剪接体蛋白与活细胞中新生前体 mRNA 的相互作用。该方法涉及在哺乳动物细胞中共表达用一种颜色标记的靶前体 mRNA 和用另一种颜色标记的剪接体蛋白。通过光漂白后荧光恢复（FRAP）或光激活来估计蛋白质的扩散系数以及其与前体 mRNA 的结合和解离速率。
