Rakefet Ben-Yishay 1 Amir Mor 1 Amit Shraga 1 Asaf Ashkenazy 1 Yuval Garini 2 Yaron Shav-Tal 1
1The Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
2Department of Physics, Bar-Ilan University, Ramat-Gan, Israel

Export of mRNA from the cell nucleus is one of the pillars of the gene expression pathway in eukaryotes. After transcription, mRNA binding proteins (RBPs), some of which are directly involved in the export process, associate with the transcript to create an mRNP. The protein composition of the mRNP changes during export. Key factors such as Nuclear Export Factor 1 (NXF1/Tap/Mex67), Aly, and the DEAD-Box protein Dbp5 are needed to execute the export process. Aly is recruited to the mRNA by the exon junction complex (EJC), and together with UAP56 and THOC5, are responsible for the binding of NXF1/Tap to the mRNA. NXF1/Tap can bind to several Nups at the nuclear pore complex (NPC) and mediate the transport of the mRNA through the pore. We knocked down Nxf1/Tap and followed the dynamics of single mRNPs in living cells. We found that mRNPs could bind but not translocate through the NPC. In contrast, during knockdown of Nup153, mRNPs could not bind nor translocate. This suggested that mRNA binding to the NPC does not require NXF1/Tap, whereas mRNA passage through NPC and release into the cytoplasm does. In accordance, super-resolution microscopy showed that NXF1/Tap was enriched on the cytoplasmic side of the NPC. We next found that NXF1/Tap was continuously present at all NPCs and under all conditions tested, namely, even during transcription inhibition, mRNA export inhibition and RNase treatment. Moreover, a NXF1/Tap mutant that cannot bind mRNA was also present at the NPC under regular conditions. These findings indicated that NXF1/Tap is consistently occupying positions within the NPC. To test this we wanted to be able to differentiate at high-resolution between the functional interactions occurring on the nuclear side of the nuclear pore complex (NPC), the inner channel of the pore, and the cytoplasmic side. To this end we implemented a FLIM-FRET approach (FRET measurements using fluorescence lifetime measurements) that enabled the detection and measurements of specific interactions taking place between NXF1/Tap and Nups in individual NPCs, in intact cells. We demonstrate the discrimination between specific interactions under regular conditions or when mRNA export is blocked, and present a map of NXF1/Tap interactions within the NPC together with the characterization of interactions involved in mRNA export.

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