Regulation of the Dbp5 ATPase cycle in mRNP remodeling at the nuclear pore: a lively new paradigm for DEAD-box proteins

Abstract

It is commonly assumed that all DEAD-box ATPases function via a shared mechanism, since this is the case for the few proteins characterized thus far. Hodge and colleagues (pp. 1052-1064) and colleagues (pp. 1065-1077) now describe a novel model for Dbp5's ATPase cycle in mRNA (messenger RNA)/protein complex (mRNP) remodeling during nuclear export. Notably, unlike other DEAD-box proteins, Dbp5 uses a conformational change distinct from ATP hydrolysis for its activity and requires an ADP release factor to reset its ATPase cycle. Dbp5 is a canonical DEAD-box protein with a novel mechanismDbp5 is one of 25 DEAD-box RNA-dependent ATPases in budding yeast. One or more DEAD-box proteins are associated with every major step of RNA processing (for recent reviews, see Cordin et al. 2006;Jarmoskaite and Russell 2010). This family of proteins is characterized by nine conserved motifs (including the eponymous DEAD motif), which contact ATP and RNA (Fig. 1A). The conserved motifs fold into two RecA-like domains connected by a flexible linker (Fig. 1B). These domains are loosely associated when nucleotide-free, but form a binding pocket for ATP and a binding surface for RNA that are each contacted by conserved residues within the characteristic motifs. Although only a handful of structures exist for DEAD-box proteins, the RecA-like domains in different proteins interact with nucleotides and RNA very similarly (Caruthers et al. 2000;Cheng et al. 2005;Andersen et al. 2006;Sengoku et al. 2006;. DEAD-box proteins accomplish a wide range of cellular tasks, including remodeling or stabilizing structured RNA, unwinding short helical regions of RNA, stabilizing protein complexes, and directly removing proteins bound to RNA. The functional specificity of individual proteins in the DEAD-box family is due at least in part to their unique N-terminal and C-terminal sequences flanking the two RecA-like domains and their interactions with regulatory protein cofactors.Several DEAD-box proteins from various organisms have been well characterized, and all function via a similar mechanism (Hilbert et al. 2009). The DEAD-box ATPase cycle begins in the absence of nucleotides and RNA with the two RecA-like domains in an open conformation. Next, ATP binding and RNA binding occur in a cooperative manner. When bound to ATP, DEAD-box proteins have a tight affinity for RNA. ATP binding is sufficient for many DEAD-box proteins to unwind short duplex RNA. Structures of RNA-bound DEAD-box proteins show a sharp kink in the backbone of the ssRNA that is incompatible with A-form helical RNA. ATP hydrolysis is not necessary for the unwinding activity of several DEAD-box proteins, which is still possible in the presence of certain nonhydrolyzable ATP analogs. However, full activity seems to require ATP hydrolysis itself, but not release of the inorganic phosphate (P i ) from the hydrolyzed ATP. Once P i is released, the ADP-bound DEAD-box protein has a low affinity for RNA and dissociates from it. Finally, ADP dissociates from the DEAD-box protein, which is...

Nuclear export of single native mRNA molecules observed by light sheet fluorescence microscopy

“…Interestingly, the overlay indicated an accumulation of positions in the basket region and adjacent entry of the central framework of the NPC. Thus, the rate-limiting step of the translocation process is probably located in that domain and not at the cytoplasmic NPC face, where the mRNA is remodeled by the RNA helicase Dbp5 (4,27,28). An important cofactor of Dbp5 is Gle1, which is also tethered to the NPC via a specific NUP interaction.…”

“…An important cofactor of Dbp5 is Gle1, which is also tethered to the NPC via a specific NUP interaction. Gle1 binds Dbp5 together with soluble Inositol-6-phosphate, IP 6 , which might trigger ATP hydrolysis (4,29). It was postulated that this reaction is the rate-limiting step in the Dbp5 ATPase cycle (29).…”

“…This process takes place at the cytoplasmic interface of the NPC, and presumably goes along with unloading of transport receptors from the mRNA. After remodeling, the mRNA cannot reenter the NPC and is released into the cytoplasm (2,4). The most detailed analyses of mRNA export were accomplished by electron microscopy (EM) using C. tentans salivary gland cells (5,6).…”

Dbp5, Gle1-IP6and Nup159

“…28, [30][31][32] Here we discuss how the data impact each model, and propose a role for IP 6 -bound Gle1 in promoting loading of ATP onto Dbp5 as a key step in the mechanism, with RNA binding to Dbp5 linked to Gle1-IP 6 release.…”

mRNA Biogenesis in the Nucleus and Its Export to the Cytoplasm

“…DEAD-box RNA helicases conjugate their ATPase activities to their helicase activities. The ATP-bound form of Dbp5, "closed form", sandwiches RNA, but the ADP-bound form, "open form", releases RNA (Ledoux and Guthrie, 2011). Moreover, only the ADP-bound Dbp5 can remove Nab2 from mRNA, whereas ATP-bound Dbp5 does not.…”