Elucidating the Mechanism of mRNA Export Regulation by the Nuclear Pore Complex

Abstract

The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in eukaryotic cells, and it facilitates and regulates the bidirectional transfer of molecules between the nucleus and cytoplasm. One of the essential functions of the NPC is to directly regulate the export of mature mRNAs, but the mechanism is not well understood, especially in humans. Export of mRNA is completed at the cytoplasmic side of the NPC, where ATPase activity of the DEAD-box helicase DDX19 is specifically activated by the NPC components Gle1, Nup42, and Nup214. The mRNA export factor Gle1 is an essential nucleoporin, and Gle1 dysfunction has been linked to human diseases. We show that the Gle1-Nup42 interaction is highly conserved with X-ray crystal structures and that the thermostability of Gle1 is highly dependent on Nup42. We also find that disease-linked mutants of Gle1 show strongly altered thermostability. Analysis of DDX19 steady-state ATPase activity reveals a novel mode of activation by Gle1 in humans. Structural studies of DDX19 complement the biochemical characterization of DDX19, and we have proposed a working model for the DDX19 catalytic cycle. An outstanding question remains in this model, as the details of DDX19-mediated removal of the export factor NXF1-NXT1 from mRNA are unknown. We describe a method for large-scale purification of retroviral constitutive transport element (CTE) RNA, which hijacks NXF1-NXT1 to be exported through the NPC, for X-ray crystallographic studies of a NXF1-NXT1-CTE complex. Together, these results provide a detailed structural and functional description of mRNA export regulation by the NPC and a framework for understanding the molecular basis of human disease linked to Gle1 and viral proliferation.