Summary Nuclear pore complexes (NPCs) are ~100 MDa transport channels assembled from multiple copies of ~30 nucleoporins (Nups). One third of these Nups contain phenylalanine-glycine (FG)-rich repeats forming a diffusion barrier, which is selectively permeable for nuclear transport receptors that interact with these repeats. Here, we identify an additional function of FG-repeats in the structure and biogenesis of the yeast NPC. We demonstrate that GLFG-containing FG-repeats directly bind to multiple scaffold Nups in vitro and act as NPC targeting determinants in vivo. Furthermore, we show that the GLFG repeats of Nup116 function in a redundant manner with Nup188, a non-essential scaffold Nup, to stabilize critical interactions within the NPC scaffold needed for late steps of NPC assembly. Our results reveal a previously unanticipated structural role for natively unfolded GLFG-repeats as velcro to link NPC subcomplexes, and thus add a new layer of connections to current models of the NPC architecture.

Single-particle imaging in budding yeast demonstrates that mRNP export is fast (∼200 ms) and that mRNPs are retained at NPCs and undergo retrograde transport in a mex67-5 mutant, proving an essential role for Mex67p in directional mRNP transport.

The RNA export factor Mex67 is essential for the transport of mRNA through the nuclear pore complex (NPC) in yeast, but the molecular mechanism of this export process remains poorly understood. Here, we use quantitative fluorescence microscopy techniques in live budding yeast cells to investigate how Mex67 facilitates mRNA export. We show that Mex67 exhibits little interaction with mRNA in the nucleus and localizes to the NPC independently of mRNA, occupying a set of binding sites offered by FG repeats in the NPC. The ATPase Dbp5, which is thought to remove Mex67 from transcripts, does not affect the interaction of Mex67 with the NPC. Strikingly, we find that the essential function of Mex67 is spatially restricted to the NPC since a fusion of Mex67 to the nucleoporin Nup116 rescues a deletion of MEX67. Thus, Mex67 functions as a mobile NPC component, which receives mRNA export substrates in the central channel of the NPC to facilitate their translocation to the cytoplasm.

The transport of messenger RNAs (mRNAs) from the nucleus to cytoplasm is an essential step in the gene expression program of all eukaryotes. Recent technological advances in the areas of RNA-labeling, microscopy, and sequencing are leading to novel insights about mRNA biogenesis and export. This includes quantitative single molecule imaging (SMI) of RNA molecules in live cells, which is providing knowledge of the spatial and temporal dynamics of the export process. As this information becomes available, it leads to new questions, the reinterpretation of previous findings, and revised models of mRNA export. In this review, we will briefly highlight some of these recent findings and discuss how live cell SMI approaches may be used to further our current understanding of mRNA export and gene expression.

Selective transport across the nuclear envelope (NE) is mediated by the nuclear pore complex (NPC), a massive ∼100-MDa assembly composed of multiple copies of ∼30 nuclear pore proteins (Nups). Recent advances have shed light on the composition and structure of NPCs, but approaches that could map their organization in live cells are still lacking. Here, we introduce an in vivo method to perform nuclear radial intensity measurements (NuRIM) using fluorescence microscopy to determine the average position of NE-localized proteins along the nucleocytoplasmic transport axis. We apply NuRIM to study the organization of the NPC and the mobile transport machinery in budding yeast. This reveals a unique snapshot of the intact yeast NPC and identifies distinct steady-state localizations for various NE-associated proteins and nuclear transport factors. We find that the NPC architecture is robust against compositional changes and could also confirm that in contrast to Chlamydomonas reinhardtii, the scaffold Y complex is arranged symmetrically in the yeast NPC. Furthermore, NuRIM was applied to probe the orientation of intrinsically disordered FG-repeat segments, providing insight into their roles in selective NPC permeability and structure.