A single molecule view on Dbp5 and mRNA at the nuclear pore
A single molecule view on Dbp5 and mRNA at the nuclear pore
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DOI：
10.4161/nucl.23386
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发表时间：
2013-01
期刊：
影响因子：
3.7
通讯作者：
Tim P. Kaminski;J. Siebrasse;U. Kubitscheck
中科院分区：
文献类型：
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作者：
Tim P. Kaminski;J. Siebrasse;U. Kubitscheck
近年来揭示了细胞内mRNA加工的许多分子细节。然而，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，尚无法在体内进行检测。问题是在不干扰mRNA分子固有行为的情况下观察它们。我们使用了一种基于蛋白质的标记方法来可视化活的Chironomus tentans唾液腺细胞中的单个天然mRNA，这是几十年来研究mRNA生命周期的标志性系统。重组hrp36是哺乳动物hnRNP A1的C. tentans同源物，荧光标记并微注射到活细胞中，将其整合到新生的mRNPs中。利用定制的薄片荧光显微镜，以高空间和时间分辨率观察单个mRNPs的核内轨迹，包括它们的NPC通道。我们分析了mRNP输出的动力学和动力学，并通过测量NPC中单个Dbp5的转化动力学开始研究其机制和调控。
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.