Structural and functional analysis of the interaction between the nucleoporin Nup214 and the DEAD-box helicase Ddx19

Abstract

Key steps in the export of mRNA from the nucleus to the cytoplasm are the transport through the nuclear pore complex (NPC) and the subsequent remodeling of messenger RNA-protein (mRNP) complexes that occurs at the cytoplasmic side of the NPC. Crucial for these events is the recruitment of the DEAD-box helicase Ddx19 to the cytoplasmic filaments of the NPC that is mediated by the nucleoporin Nup214. Here, we present the crystal structure of the Nup214 N-terminal domain in complex with Ddx19 in its ADPbound state at 2.5 Å resolution. Strikingly, the interaction surfaces are not only evolutionarily conserved but also exhibit strongly opposing surface potentials, with the helicase surface being positively and the Nup214 surface being negatively charged. We speculate that the positively charged surface of the interacting ADP-helicase binds competitively to a segment of mRNA of a linearized mRNP, passing through the NPC on its way to the cytoplasm. As a result, the ADP-helicase would dissociate from Nup214 and replace a single bound protein from the mRNA. One cycle of protein replacement would be accompanied, cooperatively, by nucleotide exchange, ATP hydrolysis, release of the ADP-helicase from mRNA and its rebinding to Nup214. Repeat of these cycles would remove proteins from a mRNP, one at a time, akin to a ratchet mechanism for mRNA export.crystal structure ͉ immunofluorescence localization ͉ mRNA export ͉ mutagenesis ͉ Nup214-helicase complex

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

“…The helicase core of Dbp5/Ddx19 is flanked by an Nterminal extension that appears to contribute to nucleotide binding (Collins et al, 2009;Fan et al, 2009;Napetschnig et al, 2009;von Moeller et al, 2009). A homologous region is also found in Ddx25.…”

“…In addition to direct and indirect contacts to the nucleotides, the conserved motifs from both RecA-like domains form an intricate interaction network around the ATPase site. In some cases, the isolated N-terminal RecA-domain interacts (weakly) with nucleotides (Rudolph et al, 2006;Fan et al, 2009;Napetschnig et al, 2009), whereas no interaction with nucleotides was detected for the YxiN N-terminal RecAlike domain (Karow et al, 2007). The interaction of the nucleotide with both domains provides the link to its influence on RNA binding and remodeling.…”

“…In Ddx19, the a-helix N-terminal to the helicase core is inserted in the cleft of the helicase core in the presence of ADP (Collins et al, 2009). When ADPNP and ssRNA are bound, the Ddx19 core adopts the closed conformation, and the N-terminal helix is packed onto the surface of the C-terminal RecA-like domain, whereas it interacts with the N-terminal RecA-like domain in the structure of Ddx19 with its interaction partner Nup214 (Napetschnig et al, 2009). Deletion experiments point towards a regulatory role of the Ddx19 N-terminal extension on Ddx19 activity (Collins et al, 2009).…”

“…When overall RNA affinities are determined, nucleotide-dependent substrate binding properties of the core will thus be masked by the nucleotideindependent high RNA affinity of the flanking domain, and it is crucial to dissect the individual contributions. Recent evidence suggests that additional domains can also influence nucleotide binding (Collins et al, 2009;Fan et al, 2009;Napetschnig et al, 2009;von Moeller et al, 2009), see also the section on 'modulation of the helicase core activity by interacting partners and flanking domains'.…”

Hooking She3p onto She2p for myosin-mediated cytoplasmic mRNA transport

“…In the nucleus, She2p is thought to associate primarily with zipcoded mRNA with the nuclear protein Loc1p and Puf6p (an mRNA-binding protein that binds to 3′ UTR) (18)(19)(20)(21). However, most nuclear messenger ribonucloprotein particles are remodeled by helicases either within the nucleus or, during nuclear export, by helicase Dbp5p sitting on guard at the cytoplasmic aspect of the nuclear pore complex (22). In the cytoplasm, She2p has several partners, such as She3p, zipcode, and Puf6p (10-13, 19, 23, 24).…”