Unravelling mRNA export

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Unravelling mRNA exportCharles N. Cole and John J. Scarcelli

Precise spatial regulation of mRNA remodelling is crucial for proper gene expression. One way this is accomplished is through activation of the DEAD-box helicase Dbp5 at the cytoplasmic fibrils of the nuclear-pore complex, by the nucleoporin Gle1 and the soluble inositol polyphosphate InsP6.

Throughout gene expression, mRNAs are asso-ciated with proteins in complexes called mes-senger ribonucleoprotein particles (mRNPs). Several mRNA-binding proteins are recruited to the nascent mRNA during transcription and nuclear processing events. Although some have roles in RNA processing, others function in later steps in gene expression, such as export1. mRNPs move to the cytoplasm through nuclear pore complexes (NPCs), large protein assem-blies (relative molecular weight (Mr) of approx-imately 120,000 K in metazoan cells) that are the only channels for transport between the nucleus and the cytoplasm.

Completion of pre-mRNA processing generates export-competent mRNPs, the structure of which facilitates efficient move-ment through NPCs. Although prema-ture remodelling would prevent or impede mRNA export, newly exported mRNPs are not structured optimally for translation, therefore remodelling is required before protein synthesis can occur. How precise spatial control of this process is achieved has been one of the most important unan-swered questions concerning mRNA export. On pages 668 and 711 of this issue, Weirich et al.2 and Alcázar-Román et al.3 show that in yeast, the enzymatic machinery predicted to mediate remodelling during export is acti-vated through critical interactions that occur at precisely the proper location — the NPC

filaments located on the cytoplasmic side of the central channel of the NPC.

The key factors in this process are: Dbp5, a member of the DEAD-box family of proteins; Gle1, a component of the cytoplasmic fila-ments; and InsP6, one of many small molecule phosphoinositides found in cells. Previous studies indicated that all three are important for mRNA export but a mechanistic understand-ing of how they affect this process was lacking. Cells contain dozens of DEAD-box proteins and one or more participate in each step of mRNA metabolism4. DEAD-box proteins are ATPases that harness energy from ATP hydrolysis to unwind short double-stranded regions of RNA or to dissociate bound proteins from RNA, although the activities of only a few DEAD-box proteins have been examined4.

Dbp5 is highly conserved among all eukary-otic organisms5,6. In Chironomous tentans, immunoelectron microscopy shows that Dbp5 can be detected near the 5′ end of mRNAs when they are nascent chains and remains there until after the mRNA has been exported to the cyto-plasm7. Dbp5 shuttles between the nucleus and cytoplasm, and at steady state, is concentrated on the cytoplasmic filaments of the NPC5,8. Although Dbp5 had been predicted to medi-ate remodelling of mRNP at this location, the mechanism that regulates its activity had not been defined. Gle1 is one of the nucleoporins of these filaments.

The studies in this issue2,3 show that Gle1 and InsP6 regulate Dbp5 activity. Previously, Gle1 mutants have been shown to have strong defects in mRNA export. Moreover, the meta-bolic pathway that produces InsP6 was discov-ered in a genetic screen for factors that mediate

Gle1 function9. At the time, this was surprising as the cellular roles of highly phosphorylated inositides were unknown. Although not strictly essential for yeast-cell viability, the importance of InsP6 production in mRNA export was fur-ther underscored by detailed genetic studies that found enhanced lethality in cells carrying mutations affecting Dbp5, and the nucleop-orins with which it interacts, if the cells were unable to produce InsP6 (ref. 10).

Weirich et al.2 and Alcázar-Román et al.3 both report that the known RNA-dependent ATPase activity of Dbp5 is stimulated dramati-cally in vitro by an interaction with Gle1, and that even greater stimulation occurs when InsP6 is also present. Similarly to many DEAD-box proteins, the ATPase activity of Dbp5 alone is low and for maximal activity requires a con-centration of ATP that may be above what is found in cells under some growth conditions. Overall, the interaction of Gle1 and InsP6 with Dbp5 substantially reduces the concentration of ATP and RNA needed for optimal activa-tion, and the net result is that the catalytic effi-ciency of Dbp5 is increased several hundred fold. Whereas Alcázar-Román et al.3 used full-length recombinant Gle1, Weirich et al.2 used the carboxy-terminal half of the protein, and this probably accounts for their need to use several-fold more InsP6 than Alcázar-Román et al.3 to achieve half-maximal stimulation.

In addition to the in vitro assays, these papers use a variety of genetic approaches that further link InsP6, Gle1 and Dbp5. By targeting highly conserved residues of Gle1, Weirich et al.2 iso-lated a mutant of Gle1 (Gle1R287A) that was sta-ble, properly localized to NPCs, but was unable to stimulate Dbp5 in vitro. This mutant strain

Charles N. Cole is in the Departments of Biochemistry and Genetics, Dartmouth Medical School, Hanover, NH 03755, USA. John J. Scarcelli is in the Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.e-mail: [email protected]; [email protected]

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also showed a reduced ability to export mRNA. Whereas wild-type Dbp5 and Gle1 interacted strongly in vivo, as measured by a yeast two-hybrid assay, the Gle1R287A mutant protein did not interact with Dbp5.

In studies to identify suppressors of the growth defect of the ipk1∆ nup42∆ strain (Nup42 is a nucleoporin that binds Gle1 at NPC cytoplasmic filaments11 and Ipk1 is the final enzyme required for synthesis of InsP6 from inositol 1,4,5-trisphosphate; Ins(1,4,5)P3; ref. 9), Alcázar-Román et al.3 identified DBP5 as a high-copy suppressor and Weirich et al.2 iso-lated dominant alleles of both DBP5 and GLE1. In two-hybrid assays, these dominant mutant proteins interacted much more strongly in the absence of InsP6 than wild-type Dbp5 and Gle1 did. Because InsP6 probably has an allosteric role in the Dbp5–Gle1 interaction, it makes

sense that mutants of Dbp5 and Gle1 may exist that would bypass the need for InsP6. When the enzymatic properties of the dominant-mutant proteins were examined in vitro, they stimulated the ATPase activity of Dbp5 equally well, irre-spective of whether InsP6 was present. These conclusions are also supported by previous genetic studies of York et al.9 showing that PLC1 overexpression, which dramatically increases all inositol polyphosphate levels, rescues a gle1 mutant only if Ipk1 is present to produce InsP6. Both Alcázar-Román et al.3 and Weirich et al.2 show that Gle1 binds InsP6 and that stimulation of Dbp5 by InsP6 requires Gle1.

Dbp5 was previously shown to con-tact the NPC primarily through its inter-action with the amino-terminal domain of Nup159 (refs 8, 12). Deletion of this domain dramatically reduces the amount of

Dbp5 detected at NPCs, indicating that the N-terminus of Nup159 serves as a docking site for Dbp5 (Fig. 1). Although nup159∆N cells are temperature-sensitive for growth and have very severe defects in mRNA export, overexpres-sion of DBP5 rescues these defects completely, even at elevated temperature. In contrast, over-expression of DBP5 does not overcome the defects of a temperature-sensitive gle1 mutant strain, suggesting that the role of Gle1 in regu-lating Dbp5 is different from that of Nup159 (ref. 8). Biochemical studies in the two papers in this issue support this model, as Gle1, but not Nup159, could stimulate the ATPase activ-ity of Dbp5 in vitro.

Several important questions remain. Although the ATPase activity of Dbp5 is essen-tial, what does Dbp5 do in vivo? Previous stud-ies were able to demonstrate an RNA unwinding

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Figure 1 Dbp5 is selectively activated on the cytoplasmic fibrils of the NPC. (a) In the nucleus, proteins, including the DEAD-box protein Dbp5, are loaded on to mRNAs during transcription and pre-mRNA processing. This includes the cap-binding complex, shown in brown. Some of these proteins are required for translocation of the mRNP through the nuclear pore. When the mRNP reaches the cytoplasmic side of the pore, Dbp5 interacts with the N-terminus of Nup159, which is present on the cytoplasmic filaments and is thought to serve as a docking site. At this point, Dbp5 becomes enzymatically active due to interactions with the essential mRNA export

factor Gle1, a component of the cytoplasmic fibrils, and the soluble inositol polyphosphate InsP6. Activation of the ATPase activity of Dbp5 allows it to remove mRNA-binding proteins required for nuclear transport from the mRNP, so these can return to the nucleus for another round of export. (b) A magnified view of Dbp5 acting to remove proteins from an mRNP when bound to the cytoplasmic NPC fibrils. Nup42 helps to anchor Gle1 to the fibrils and Gfd1 is a non-essential protein that interacts with Dbp5, Gle1 and Nup42. Through the activity of Dbp5, the mRNP can move to the cytoplasm where the mRNA can then be translated.



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activity for Dbp5 only in the presence of a cel-lular extract6. Weirich et al.2 present evidence that Dbp5 alone has an ATP-dependent RNA unwinding activity. However, a twofold molar excess of Dbp5 was required to unwind a short double-stranded RNA substrate and the effect of Gle1 and InsP6 on this activity was not exam-ined. Thus, additional studies will be required to determine whether Dbp5 unwinds double-stranded regions of mRNA in vivo or dissociates proteins from the mRNP, or both, and what role stimulatory factors play in its activity. The stud-ies here suggest that Dbp5 must interact with Gle1 to trigger its activation. Does Dbp5 act enzymatically only when in contact with Gle1 at the cytoplasmic face of the NPC? Perhaps this is the case, but given the prolonged association of Dbp5 with mRNPs, there may be additional cofactors working at other steps of mRNA metabolism that also activate Dbp5 and provide different specificity.

What proteins does Dbp5 remove when it interacts with the cytoplasmic NPC filaments? Mex67 functions as a receptor for mRNA export and, as a heterodimer with Mtr2, can bind to both mRNPs and nucleoporins, thereby facilitating mRNA export13. This makes Mex67 and Mtr2 reasonable candidates for removal by Dbp5. Recently, it was demonstrated that muta-tion of Dbp5 leads to increased association of Mex67 with mRNPs, suggesting that Dbp5 might remove Mex67 in vivo14. A demonstra-tion that Dbp5, Gle1 and InsP6 can do this in vitro would be an important step forward in defining the precise role of Dbp5 at the NPC.

However, this may be difficult to reca-pitulate in vitro. Only two DEAD-box pro-teins, Ded1 and NPH-II, have been shown to remove bound proteins from RNA in vitro15. Importantly, both proteins were active on

one of the model substrates used but only one could act on the other, indicating that different DEAD-box proteins have distinct substrate specificities. In addition, the inter-action of Mex67 with mRNA is thought to be mediated by other proteins, therefore pre-paring a proper substrate for these in vitro studies is challenging. Furthermore, the NPC could provide an essential structural platform for this process. Is Mex67 the only protein removed from mRNPs at the NPC by Dbp5, or are there others? It will also be important to determine how many proteins Dbp5 can displace per molecule of ATP hydrolysed and whether a single molecule of Dbp5 remains associated with an mRNP for multiple cycles of ATP hydrolysis and mRNP remodelling.

These two papers provide insight into the energy source and mechanism used for trans-location of mRNPs through NPCs. The protein export machinery, including Ran and the kary-opherins, does not have a direct role in export of mRNPs. An important consideration here is the size of mRNPs, which are very much larger than protein–karyopherin complexes and are almost certainly too large to move through the NPC efficiently by diffusion. In contrast with DNA helicases, which are highly processive and able to move along DNA for thousands of bases, different DEAD-box proteins have either limited or no processivity. Whether Dbp5 can act processively is not known. When DEAD-box proteins act processively, they move along RNA in the manner of a molecular motor16. The intriguing possibility for Dbp5 is that it would function to remove bound proteins while itself bound to the NPC. In this model, the mRNP, not the motor, is the mobile component, mov-ing vectorially from the nucleus to the cyto-plasm through the action of Dbp5. This could

occur either through the processive action of an individual Dbp5 molecule or the coordinate action of more than one Dbp5, perhaps bound to adjacent cytoplasmic NPC filaments or to each other.

The enzymatic analyses of Weirich et al.2

show that the interaction between Dbp5 and RNA in vitro is very brief and if this is true for Dbp5–mRNP interactions in vivo then mRNA export may be mediated by the coordinated action of multiple Dbp5 molecules acting sequentially to pull the mRNP through the NPC. Determining whether this model is cor-rect is a major challenge due to the complexity of the components and the difficulty of creating an in vitro assay. Hopefully, steady progress in defining the biochemical properties of Dbp5, coupled with further insights gained from genetic approaches, will clarify the mechanism of mRNP export.

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Unravelling mRNA export