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Nuclear export of single native mRNA molecules observed by light sheet fluorescence microscopy

Siebrasse, Jan Peter, Kaminski, Tim, Kubitscheck, Ulrich
Proceedings of the National Academy of Sciences of the United States of America 2012 v.109 no.24 pp. 9426-9431
Chironomus tentans, RNA helicases, RNA transport, eukaryotic cells, fluorescence, fluorescence microscopy, messenger RNA, models, nuclear membrane, nucleoporins, salivary glands
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.