Nuclear export of single native mRNA molecules observed by light sheet fluorescence microscopy
Nuclear export of single native mRNA molecules observed by light sheet fluorescence microscopy
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DOI：
10.1073/pnas.1201781109
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发表时间：
2012-05
期刊：
影响因子：
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通讯作者：
J. Siebrasse;Tim P. Kaminski;U. Kubitscheck
中科院分区：
文献类型：
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作者：
J. Siebrasse;Tim P. Kaminski;U. Kubitscheck
mRNA的核输出是真核细胞中一个关键的转运过程。为了研究它，我们用荧光hrp36、hnRNP A1同源物和荧光NTF2标记活鼠唾液腺细胞中的天然mRNP颗粒。使用薄片显微镜，我们追踪单个天然mRNA颗粒穿过核膜。观察到颗粒经常在其核面探测核孔复合物（NPC），只有25%的情况下产生实际出口。完整的导出过程耗时从65毫秒到几秒钟。观察到一个速率限制步骤，该步骤可以分配给孔的核篮，并且可能对应于实际易位之前mRNPs的重新定位和展开。对mRNA输出所必需的RNA解旋酶Dbp5分子的单荧光分析显示，Dbp5最常接近鼻咽癌细胞质表面，并显示出约55 ms的结合持续时间。我们的研究结果允许对mRNA输出的当前模型进行改进。
Nuclear export of mRNA is a key transport process in eukaryotic cells. To investigate it, we labeled native mRNP particles in living Chironomus tentans salivary gland cells with fluorescent hrp36, the hnRNP A1 homolog, and the nuclear envelope by fluorescent NTF2. Using light sheet microscopy, we traced single native mRNA particles across the nuclear envelope. The particles were observed to often probe nuclear pore complexes (NPC) at their nuclear face, and in only 25% of the cases yielded actual export. The complete export process took between 65 ms up to several seconds. A rate-limiting step was observed, which could be assigned to the nuclear basket of the pore and might correspond to a repositioning and unfolding of mRNPs before the actual translocation. Analysis of single fluorescent Dbp5 molecules, the RNA helicase essential for mRNA export, revealed that Dbp5 most often approached the cytoplasmic face of the NPC, and exhibited a binding duration of approximately 55 ms. Our results have allowed a refinement of the current models for mRNA export.