Three-Dimensional Mapping Nuclear Export of Single mRNAs with Exceptional Spatiotemporal Accuracy

Abstract

In eukaryotic cells, the flow of genetic information is regulated by selective nucleocytoplasmic transport of individual messenger RNA:protein complexes (mRNPs) through the nuclear pore complex (NPC). Each NPC, ∼200 nm in length and ∼50 nm in inner diameter, is a large assembly of multiple copies of ∼30 nucleoporins. However, the nuclear export kinetics, three-dimensional (3D) pathway and selectivity step of mRNPs transiting an individual NPCs remain poorly understood. Here we employ single-molecule fluorescence microscopy with an unprecedented spatiotemporal super-accuracy of 8 nm and 2 ms to characterize nuclear mRNP export in living cells. We find that, mRNPs exiting the nucleus are decelerated and selected at the narrowest region of the NPC, and adopt a fast-slow-fast diffusion pattern as they translocate through the NPC. 3D mapping translocation pathway for mRNPs further indicates only one third of all mRNAs successfully transits during their ∼12-ms interaction with the NPC, primarily follow the periphery on the nucleoplasmic side and in the center of the NPC without occupying central axial conduit utilized for passive diffusion of small molecules, and eventually dissociating from the cytoplasmic side.