The export of mRNA from the cell nucleus is one of the pillars of the gene expression pathway in eukaryotes. We set out to characterize interactions involved in the various stages of mRNA transition through individual nuclear pores. Using different types of mRNA export blocks together with single-molecule mRNA tracking in living human cells we could detect the regions of the NPC at which the mRNAs were stuck. Our results suggest that the initial 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 is consistently occupying positions within the cytoplasmic side of the NPC. Measurements performed within individual nuclear pores using super-resolution STED microscopy, showed the positioning of NXF1/Tap in individual pores during regular and export blockage conditions. We then could further pinpoint these interactions with specific nucleoporins using a FLIM-FRET approach for measuring protein-protein interactions inside single NPCs. Altogether, these approaches have allowed the detection and measurements in intact cells, of specific interactions taking place between NXF1/Tap and mRNAs, and between NXF1/Tap and proteins within the NPCs. We have found that specific interactions at the cytoplasmic side of the NPC are crucial for the release of the mRNA to the cytoplasm, and have identified positions within the NPC that are mechanistically fundamental for the directional flow of mRNA and NXF1/Tap from the nucleus into the cytoplasm.
