Abstract
The nuclear pore complex (NPC) controls the passage of macromolecules between the nucleus and cytoplasm, but how the NPC directly participates in macromolecular transport remains poorly understood. In the final step of mRNA export, the DEAD-box helicase DDX19 is activated by the nucleoporins Gle1, Nup214, and Nup42 to remove Nxf1•Nxt1 from mRNAs. Here, we report crystal structures of Gle1•Nup42 from three organisms that reveal an evolutionarily conserved binding mode. Biochemical reconstitution of the DDX19 ATPase cycle establishes that human DDX19 activation does not require IP6, unlike its fungal homologs, and that Gle1 stability affects DDX19 activation. Mutations linked to motor neuron diseases cause decreased Gle1 thermostability, implicating nucleoporin misfolding as a disease determinant. Crystal structures of human Gle1•Nup42•DDX19 reveal the structural rearrangements in DDX19 from an auto-inhibited to an RNA-binding competent state. Together, our results provide the foundation for further mechanistic analyses of mRNA export in humans.
Highlights
The nuclear pore complex (NPC) controls the passage of macromolecules between the nucleus and cytoplasm, but how the nuclear pore complexes (NPCs) directly participates in macromolecular transport remains poorly understood
Each NPC is composed of a ~60 MDa symmetric core that is decorated by different proteins on its nuclear and cytoplasmic faces, which are referred to as the nuclear basket and cytoplasmic filament nucleoporins, respectively (Fig. 1a)
To gain a better understanding of the molecular architecture of the nucleoporins that regulate mRNA export, we set out to reconstitute the interactions with purified, recombinant proteins
Summary
The nuclear pore complex (NPC) controls the passage of macromolecules between the nucleus and cytoplasm, but how the NPC directly participates in macromolecular transport remains poorly understood. Each NPC is composed of a ~60 MDa symmetric core that is decorated by different proteins on its nuclear and cytoplasmic faces, which are referred to as the nuclear basket and cytoplasmic filament nucleoporins, respectively (Fig. 1a). The outer rings, which serve as the attachment sites for the nuclear basket and cytoplasmic a Cytoplasmic filaments. Outer CNC f Identification of the Gle[1] binding site suggests that the unassigned cytoplasmic density adjacent to bridging Nup[155] molecules could contain Gle[1] and its binding partners. Right: zoomed view of unassigned cytoplasmic density, with Nup[155] shown in orange, Gle1N binding site colored in green, and coat nucleoporin complexes shown in yellow filaments, are structurally connected to the inner ring via bridging Nup[155] molecules. The molecular organization of the cytoplasmic filaments and nuclear basket remains poorly understood
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