Efficient NES-dependent protein nuclear export requires ongoing synthesis and export of mRNAs

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Abstract

The mechanisms regulating nuclear export of proteins are not fully understood. To investigate whether the efficiency of protein nuclear export may depend on ongoing RNA synthesis and/or mRNA nuclear export, we used a microinjection approach with a fluorescent reporter protein containing a nuclear export signal (NES) and scored protein export in human fibroblasts under conditions when the synthesis or export of mRNAs was inhibited. We show that inhibition of transcription significantly attenuated generic NES-dependent nuclear export. Furthermore, digestion of endogenous nuclear RNAs by co-microinjection of RNAse A inhibited NES-dependent nuclear export. Finally, nuclear export of the NES reporter protein was significantly inhibited in cells in which nuclear export of mRNAs had been specifically blocked by microinjection of anti-TAP antibodies or by expression of a dominant negative form of NUP160. These results demonstrate a novel role for ongoing synthesis and export of mRNAs in NES-dependent protein nuclear export.

Introduction

Transport of macromolecules between the nucleus and the cytoplasm occurs through the nuclear pore complex (NPC) [1], [2], [3]. Proteins that contain leucine-rich nuclear export signal (NES) domains are bound to and exported by CRM1, a member of the importin-β family [4], [5], [6], [7], [8]. It is thought that CRM1 binds in a RanGTP-dependent manner to the NES domain of the protein to be exported and to the FG repeats of the nucleoporins of the NPCs [9]. The CRM1-NES-RanGTP complex translocates through the NPC to the cytoplasmic side of the pore and subsequent hydrolysis of RanGTP causes the complex to disassemble releasing the NES-containing protein into the cytoplasm.

Many different classes of RNA such as mRNA, tRNA, rRNA, and UsnRNA are exported in complexes with proteins (ribonucleoprotein complexes) through defined pathways, some of which use importin-β family members as nuclear transport factors [10], [11], [12]. In addition to directing nuclear export of NES-containing proteins, CRM1 has also been shown to direct the nuclear export of UsnRNAs, rRNA, and certain mRNAs [12], [13], [14], [15], [16]. However, the export of bulk mRNA does not appear to depend on CRM1 or RanGTP [17], [18]. As mRNAs are processed in the nucleus, they become associated with many proteins and there is evidence that these processing reactions (such as splicing) are necessary for the interaction of the RNA with the export machinery [19], [20], [21], [22], [23], [24], [25]. Splicing might enhance the efficiency of mRNA export because intron-containing mRNAs have been shown to be exported more efficiently then intronless mRNAs [19]. As the nascent RNA undergoes splicing, it becomes bound to Aly, a component of the exon–exon junction complex [26], [27]. The bound Aly then interacts with TAP, the main export factor for mRNA [26], [27]. However mRNA export is most likely more complex then this because splicing is not absolutely required for the recruitment of nuclear mRNA export factors to mRNA [28], [29], [30]. For example, it has been shown that Aly can also be recruited to intronless mRNAs [29]. Also, to add to the complexity of nuclear mRNA export, Aly appears to not be essential for bulk mRNA export. Recent research has shown that other proteins such as hnRNP A1, SRp20, 9G8, ASF/SF2, and HuR can act as adaptors between mRNA and TAP independently of splicing [31], [32], [33], [34]. These data suggest that multiple adaptor proteins may link mRNPs and TAP to allow for efficient nuclear export.

Although it is clear that most RNAs need to be bound to proteins to undergo nuclear export, it is not thoroughly understood whether the export of proteins is dependent on the nuclear export of RNA. Several in vitro studies using permeabilized cells or isolated nuclear envelopes have shown that recombinant CRM1 and RanGTP are sufficient for the transport of a NES-containing substrate across the nuclear envelope via the NPC [35], [36], [37], [38]. It is thought that other proteins such as RanBP1 and NXT1 are then needed for the terminal step of export when the export complex disassociates [35]. However, in intact cells, other molecules, such as RNA, might be necessary for efficient nuclear export of NES-containing proteins. Recently, it was shown that both the binding and export of the eukaryotic elongation factor 1A by exportin-5, a member of the nuclear transport family, depends on the binding of aminoacylated tRNA to exportin-5 [39], [40]. This suggests that at least in this one case, RNA may aid nuclear export of proteins.

In this study, we examined the dependency of generic NES-mediated protein export on the synthesis and export of mRNAs. We hypothesized that inhibition of mRNA synthesis might block NES- and CRM1-mediated protein nuclear export. Furthermore, proteins to be exported may associate with exporting RNA complexes. To test these hypotheses, we analyzed the ability of cells to export a microinjected NES-reporter protein under conditions when the synthesis or nuclear export of mRNAs was inhibited. Our results suggest that ongoing synthesis and export of mRNA are required for efficient nuclear export of NES-containing proteins.

Section snippets

Reagents

All experiments were performed using hTert-immortalized diploid human fibroblasts. Actinomycin D, 5,6-dichloro-1-b-d-ribofuranosyl-benzimidazole (DRB), 1-(5-isoquinolinylsulfonyl)-3-methylpiperazine (H7), rabbit IgG, 70S rhodamine-dextran, and β2-microgloblulin were purchased from Sigma. RNAse A (bovine pancreas) was purchased from Roche. LMB was kindly given to us by Dr. M. Yoshida, University of Tokyo.

GST-NES-GFP protein isolation and microinjection

GST-NES-GFP and GST-NLS-GFP proteins were isolated from bacteria and purified as previously

Inhibition of transcription results in blockage of generic protein nuclear export

To study generic CRM1-mediated nuclear export of NES-containing proteins, we utilized a reporter protein (GST-NES-GFP) that was microinjected into the nuclei of cells [41]. This reporter protein consists of a GST domain for purifying the protein from bacterial cell lysates, a consensus CRM1-dependent nuclear export signal (NES), and a GFP domain for visualization using fluorescent microscopy. 70S rhodamine-dextran was co-microinjected with the NES reporter protein to mark the injected cells and

Discussion

This study demonstrates a novel connection between the nuclear export of NES-containing proteins and the synthesis and nuclear export of mRNA. This conclusion is based on our results obtained using three different approaches. First, we show that the nuclear export of a NES-containing reporter protein was significantly attenuated by agents that inhibit transcription such as UV light, actinomycin D, DRB, H7, and microinjection of anti-RNA polymerase II antibodies Fig. 1, Fig. 2. Second, digestion

Acknowledgements

We thank Drs. Roland Stauber (University of Erlangen-Nurnberg) and Douglass Forbes (University of California at San Diego) for their generous gifts of plasmids; Mike Glynn and Thomas Glover (University of Michigan) for the immortalization of the human fibroblast cells with hTert; and M. Yoshida (The University of Tokyo) for the gift of LMB. We also thank Drs. Gabriel Nuñez and Thom Saunders (University of Michigan) for technical assistance with microinjections and Thomas Glover and John Moran

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    Supplementary data associated with this article can be found, in the online version, at doi: 10.1016/j.yexcr.2004.03.051.

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