Structural and functional analysis of mRNA export regulation by the nuclear pore complex

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.

Mechanisms of nuclear mRNA export: A structural perspective

“…Structure of the human DDX19•GLE1•NUP42 complex reveals that NUP42 does not contact DDX19 directly, and causes no significant conformational change in GLE1 (Figure F) . The effect of NUP42 is suggested to be attributed to increasing GLE1 thermostability; the melting temperature of GLE1 increased from 37 to 50 °C in the presence of NUP42 . Of note, in the human DDX19•GLE1•NUP42 complex, only DDX19‐CTD is engaged in GLE1 binding, whereas Gle1 contacts both Dbp5 NTD and CTD in the yeast Dbp5•Gle1•IP 6 complex.…”

“…In the mRNA export platform, Gle1, IP 6 (inositol hexakisphosphate) and Nup42 provide spatial and temporal regulation of Dbp5 by stimulating its ATPase activity (Figure B). Dbp5 contains two RecA‐like domains (NTD and CTD) and a short N‐terminal extension (NTE).…”

“…A unique feature of the Gle1‐Dbp5 interaction is that IP 6 bridges the protein interaction at the interface of Gle1 and Dbp5‐CTD. Gle1‐mediated Dbp5 activation is conserved from yeast to humans . However, IP 6 activation of human DDX19 was only observed using recombinant DDX19 expressed in the baculovirus‐insect cell expression system which carries most of the post‐translational modification pathways present in mammalian systems, but not observed using DDX19 expressed in E. coli .…”

“…Nup42 interacts with Gle1, and they both are required for the export of heat shock mRNAs following stress . Structure of the human DDX19•GLE1•NUP42 complex reveals that NUP42 does not contact DDX19 directly, and causes no significant conformational change in GLE1 (Figure F) . The effect of NUP42 is suggested to be attributed to increasing GLE1 thermostability; the melting temperature of GLE1 increased from 37 to 50 °C in the presence of NUP42 .…”

Nuclear export mechanisms of circular RNAs: size does matter

“…A limited set of studies have also shown that aside from XPO5, the export of few precursor miRNAs occurs through XPO1 as well (10). The mRNAs have a distinct export mechanism that occurs with the help of adaptor serine/arginine rich proteins that promote the recruitment of heterodimeric mRNA export receptor NXF1-NXT1 which facilitates exit through the nuclear pore (11). Despite some available knowledge on microRNA nuclear transport, the non-coding RNA nuclear transport in general remains an under-studied field.…”