Biochemical Analysis of the Protein-Protein Interactions Involved in Karyopherin-Mediated Transport Across the Nuclear Pore Complex

Nucleocytoplasmic transport occurs through the nuclear pore complex (NPC), which in yeast is a highly symmetric ~50 MDa complex consisting of approximately 30 different proteins. Small molecules can freely exchange through the NPC, but macromolecules larger than ~40 kDa such as proteins, mRNAs, and ribosomal subunits must be aided across by shuttle proteins (karyopherins, or Kaps). Kap-mediated transport involves FG-nups, a family of NPC proteins. While much has been learned about the mechanism of nucleocytoplasmic transport, many details are still unknown; perhaps among the most important missing details is the binding kinetics of almost all the transport relevant interactions, due to significant technical challenges. The aim of this work is to analyze the protein-protein interactions involved in Kap-mediated transport across the NPC, using biochemical, biophysical, and cell biological approaches. Yeast karyopherins, model cargoes, and full-length FG-nups are enriched from bacteria, and their affinities are studied quantitatively. The presence of competitor proteins and changes in bait protein distribution are seen to effect apparent affinity of these interactions. The relevance of the in vitro Kap/NLS-cargo binding measurements is confirmed with a nucleocytoplasmic import assay that allows quantitative measurements of import to be made within single living cells. Trends observed in vitro for Kap/FG-nup interactions were consistent with ex vivo observations of interactions of transport factors with Xenopus oocyte NPCs and also with in vitro measurements of transport through a synthetic NPC-based filter. This work has suggested a role for factors such as non-specific competition in determining the kinetics and selectivity of transport

Evolution of the nucleus

Under a Creative Commons license.The nucleus represents a major evolutionary transition. As a consequence of separating translation from transcription many new functions arose, which likely contributed to the remarkable success of eukaryotic cells. Here we will consider what has recently emerged on the evolutionary histories of several key aspects of nuclear biology; the nuclear pore complex, the lamina, centrosomes and evidence for prokaryotic origins of relevant players.Work in our laboratories was supported by the following agencies, and which is gratefully acknowledged; MRC and Wellcome Trust (MR/K008749/1 and 090007/Z/09/Z respectively, to MCF), C2A Junta de Andalucia to DPD and DFG GR1642/4-1 to RG.Open Access funded by Wellcome Trust.Peer Reviewe

Nanoscale stiffness topography reveals structure and mechanics of the transport barrier in intact nuclear pore complexes

The nuclear pore complex (NPC) is the gate for transport between the cell nucleus and the cytoplasm. Small molecules cross the NPC by passive diffusion, but molecules larger than ∼5 nm must bind to nuclear transport receptors to overcome a selective barrier within the NPC1. Although the structure and shape of the cytoplasmic ring of the NPC are relatively well characterized2, 3, 4, 5, the selective barrier is situated deep within the central channel of the NPC and depends critically on unstructured nuclear pore proteins5, 6, and is therefore not well understood. Here, we show that stiffness topography7 with sharp atomic force microscopy tips can generate nanoscale cross-sections of the NPC. The cross-sections reveal two distinct structures, a cytoplasmic ring and a central plug structure, which are consistent with the three-dimensional NPC structure derived from electron microscopy2, 3, 4, 5. The central plug persists after reactivation of the transport cycle and resultant cargo release, indicating that the plug is an intrinsic part of the NPC barrier. Added nuclear transport receptors accumulate on the intact transport barrier and lead to a homogenization of the barrier stiffness. The observed nanomechanical properties in the NPC indicate the presence of a cohesive barrier to transport and are quantitatively consistent with the presence of a central condensate of nuclear pore proteins in the NPC channel

Mechanism of Nonsense-Mediated mRNA Decay Stimulation by Splicing Factor SRSF1

Summary The splicing factor SRSF1 promotes nonsense-mediated mRNA decay (NMD), a quality control mechanism that degrades mRNAs with premature termination codons (PTCs). Here we show that transcript-bound SRSF1 increases the binding of NMD factor UPF1 to mRNAs while in, or associated with, the nucleus, bypassing UPF2 recruitment and promoting NMD. SRSF1 promotes NMD when positioned downstream of a PTC, which resembles the mode of action of exon junction complex (EJC) and NMD factors. Moreover, splicing and/or EJC deposition increase the effect of SRSF1 on NMD. Lastly, SRSF1 enhances NMD of PTC-containing endogenous transcripts that result from various events. Our findings reveal an alternative mechanism for UPF1 recruitment, uncovering an additional connection between splicing and NMD. SRSF1’s role in the mRNA’s journey from splicing to decay has broad implications for gene expression regulation and genetic diseases