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A single molecule view on Dbp5 and mRNA at the nuclear pore

机译：Dbp5和mRNA在核孔处的单分子视图

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Numerous molecular details of intracellular mRNA processing have been revealed in recent years. However, the export process of single native mRNA molecules, the actual translocation through the nuclear pore complex (NPC), could not yet be examined in vivo. The problem is observing mRNA molecules without interfering with their native behavior. We used a protein-based labeling approach to visualize single native mRNPs in live salivary gland cells of Chironomus tentans, an iconic system used for decades to study the mRNA life cycle. Recombinant hrp36, the C. tentans homolog of mammalian hnRNP A1, was fluorescence labeled and microinjected into living cells, where it was integrated into nascent mRNPs. Intranuclear trajectories of single mRNPs, including their NPC passage, were observed with high space and time resolution employing a custom-built light sheet fluorescence microscope. We analyzed the kinetics and dynamics of mRNP export and started to study its mechanism and regulation by measuring the turnover-kinetics of single Dbp5 at the NPC.
机译：近年来已经揭示了细胞内mRNA加工的许多分子细节。但是，单个天然mRNA分子的输出过程，即通过核孔复合体（NPC）的实际易位，尚未在体内进行检查。问题在于观察mRNA分子而不干扰其天然行为。我们使用了一种基于蛋白质的标记方法来可视化藤天牛（Chironomus tentan）唾液腺细胞中的单个天然mRNP，这是数十年来用于研究mRNA生命周期的标志性系统。重组hrp36， C。哺乳动物hnRNP A1的丹坦同源物被荧光标记并显微注射到活细胞中，在活细胞中被整合到新生的mRNP中。使用定制的光片荧光显微镜以高空间和时间分辨率观察到单个mRNP的核内轨迹，包括其NPC通道。我们分析了mRNP出口的动力学和动力学，并开始通过测量NPC上单个Dbp5的周转动力学来研究其机制。

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