The Dbp5 cycle at the nuclear pore complex during mRNA export I: <i>dbp5</i> mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1

Hodge, Christine A.; Tran, Elizabeth; Noble, Kristen N.; Alcázar-Román, Abel R.; Ben-Yishay, Rakefet; Scarcelli, John J.; Folkmann, Andrew W.; Shav‐Tal, Yaron; Wente, Susan R.; Cole, Charles N.

doi:10.1101/gad.2041611

Cited by 101 publications

(159 citation statements)

References 60 publications

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“…In the accompanying study (Hodge et al 2011), we found that such mutants behave as predicted. Notably, the ATP hydrolysis-defective dbp5 EQ mutant, which binds to either ATP or ADP ( Fig.…”

Section: Discussionmentioning

confidence: 55%

“…Notably, the ATP hydrolysis-defective dbp5 EQ mutant, which binds to either ATP or ADP ( Fig. 2A,B), is a loss-of-function mutant in vivo (Hodge et al 2011). Further, at steady state in the presence of wild-type Dbp5, dbp5 EQ protein is not detected at the nuclear rim/NPC (Hodge et al 2011).…”

Section: Discussionmentioning

confidence: 99%

“…This suggests that ADP binding or the Dbp5-ADP-bound (Dbp5 ADP ) state is functional (Tran et al 2007). However, both ATP hydrolysis and nucleotide binding are required for in vivo function, as dbp5 mutants lacking either function are recessive lethal (Hodge et al 2011). It is unknown how the in vitro ADP-dependent remodeling activity is linked to the Dbp5 mechanism for ATP hydrolysis in a cell.…”

mentioning

confidence: 99%

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The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1

Noble¹,

Tran²,

et al. 2011

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Essential messenger RNA (mRNA) export factors execute critical steps to mediate directional transport through nuclear pore complexes (NPCs). At cytoplasmic NPC filaments, the ATPase activity of DEAD-box protein Dbp5 is activated by inositol hexakisphosphate (IP 6 )-bound Gle1 to mediate remodeling of mRNA-protein (mRNP) complexes. Whether a single Dbp5 executes multiple remodeling events and how Dbp5 is recycled are unknown. Evidence suggests that Dbp5 binding to Nup159 is required for controlling interactions with Gle1 and the mRNP. Using in vitro reconstitution assays, we found here that Nup159 is specifically required for ADP release from Dbp5. Moreover, Gle1-IP 6 stimulates ATP binding, thus priming Dbp5 for RNA loading. In vivo, a dbp5-R256D/ R259D mutant with reduced ADP binding bypasses the need for Nup159 interaction. However, NPC spatial control is important, as a dbp5-R256D/R259D nup42D double mutant is temperature-sensitive for mRNA export. Further analysis reveals that remodeling requires a conformational shift to the Dbp5-ADP form. ADP release factors for DEAD-box proteins have not been reported previously and reflect a new paradigm for regulation. We propose a model wherein Nup159 and Gle1-IP 6 regulate Dbp5 cycles by controlling its nucleotide-bound state, allowing multiple cycles of mRNP remodeling by a single Dbp5 at the NPC.

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“…In the accompanying study (Hodge et al 2011), we found that such mutants behave as predicted. Notably, the ATP hydrolysis-defective dbp5 EQ mutant, which binds to either ATP or ADP ( Fig.…”

Section: Discussionmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1

Noble¹,

Tran²,

et al. 2011

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“…2b), elements required for ATP binding and hydrolysis, respectively 21,22 . As FRH is essential for the viability of Neurospora 7 , we expressed mutant and WT versions of FLAG-tagged FRH in an frh þ background.…”

Section: Resultsmentioning

confidence: 99%

The RNA helicase FRH is an ATP-dependent regulator of CK1a in the circadian clock of Neurospora crassa

et al. 2014

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The Neurospora clock protein FRQ forms a complex with casein kinase 1a (CK1a) and FRH, a DEAD box-containing RNA helicase with a clock-independent essential function in RNA metabolism. In the course of a circadian period, FRQ is progressively hyperphosphorylated and eventually degraded. Timed hyperphosphorylation of FRQ is crucial for timekeeping of the clock. Here we show that the ATPase activity of FRH attenuates the kinetics of CK1a-mediated hyperphosphorylation of FRQ. Hyperphosphorylation of FRQ is strictly dependent on site-specific recruitment of a CK1a molecule that is activated upon binding. The FRH ATPase cycle regulates the access of CK1a to phosphorylation sites in FRQ in cis, suggesting that FRH is an ATP-dependent remodelling factor acting on the protein complex. We show that the affinity of CK1a for FRQ decreases with increasing FRQ phosphorylation, suggesting functional inactivation of FRQ in the negative feedback loop of the circadian clock before and independent of its degradation.

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“…Binding of Nup159 blocks the RNA-binding site of Dbp5 (REFS 17,121) (FIG. 5) and this is required for the release of ADP, adding a new level of DEAD box protein regulation by partner proteins 123,124 . So far, such an ADP-release factor has not been described for any other DEAD box protein, but it may not be unique to Dbp5.…”

Section: Nuclear Specklesmentioning

confidence: 99%

From unwinding to clamping — the DEAD box RNA helicase family

Linder

Jankowsky

2011

Nat Rev Mol Cell Biol

956

1,069

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Nearly all aspects of RNA metabolism, from transcription and translation to mRNA decay, involve RNA helicases, which are enzymes that use ATP to bind or remodel RNA and RNA-protein complexes (ribonucleoprotein (RNP) complexes) 1 . RNA helicases are found in all three domains of life, and many viruses also encode one or more of these proteins 2,3 . Together with the structurally related DNA helicases that function in replication, recombination and repair, the RNA helicases are classified into superfamilies and families, based on sequence and structural features 3,4 . DEAD box proteins form the largest helicase family, with 37 members in humans and 26 in Saccharomyces cerevisiae 3 , and are characterized by the presence of an Asp-Glu-Ala-Asp (DEAD) motif.DEAD box helicases have central and, in many cases, essential physiological roles in cellular RNA metabolism 5 (FIG. 1). The proteins generally function as part of larger multicomponent assemblies, such as the spliceosom e or the eukaryotic translation initiation machinery 6 . Mutations and deregulation of several DEAD box proteins have been linked to disease states, including cancer 7 . Although all DEAD box proteins contain a structurally highly conserved core with conserved ATP-binding and RNA-binding sites, different proteins have been associated with diverse and seemingly unrelated functions, including the disassembly of RNPs, chaperoning during RNA folding and even stabil ization of protein complexes on RNA 5,6 . How these highly conserved proteins fulfil such an array of different functions has been a long-standing question.Research has now started to illuminate the molecular basis for this functional diversity. In this Review, we summarize how structural data, together with biochemical and biophysical studies, have revealed unexpected modes by which these proteins function. We also discuss how novel molecular and cell biological approaches have better defined the cellular roles of DEAD box helicases. We outline our current view on common structural and mechan istic themes that have emerged, and on physical models of how these 'unusual' RNA helicases work.Structures of DEAD box RNA helicases A number of structural studies have universally shown that members of the DEAD box family contain a highly conserved helicase core that harbours the binding sites for ATP and RNA 3,4,8 . The core is surrounded by variable auxiliary domains, which are thought to be critical for the diverse functions of these enzymes.Structure of the helicase core. DEAD box proteins belong to helicase superfamily 2 (SF2) 2 . Similarly to all SF2 helicases, DEAD box proteins are built around a highly conserved helicase core of two virtually identical domains that resemble the bacterial recombination protein recombinase A (RecA) 3,4,8 (FIG. 2a,b). Within this helicase core, at least 12 characteristic sequence motifs are located at conserved positions (FIG. 2a,b). Some of these motifs are conserved across the entire SF2 family, whereas others are found only in the DEAD box family 3 ....

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The Dbp5 cycle at the nuclear pore complex during mRNA export I: dbp5 mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1

Cited by 101 publications

References 60 publications

The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1

The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1

The RNA helicase FRH is an ATP-dependent regulator of CK1a in the circadian clock of Neurospora crassa

From unwinding to clamping — the DEAD box RNA helicase family

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