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

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