The Chironomus tentans translation initiation factor eIF4H is present in the nucleus but does not bind to mRNA until the mRNA reaches the cytoplasmic perinuclear region

Petra Björk1, Göran Baurén2, Birgitta Gelius3, Örjan Wrange3 and Lars Wieslander1,*

1 Department of Molecular Biology and Functional Genomics, Stockholm University, SE-106 91 Stockholm, Sweden
2 Global Genomics, Tomtebodavägen 21, SE-171 77 Stockholm, Sweden
3 Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, SE-171 77 Stockholm, Sweden

* Author for correspondence (e-mail: lars.wieslander{at}molbio.su.se)

Accepted 11 July 2003


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In the cell nucleus, precursors to mRNA, pre-mRNAs, associate with a large number of proteins and are processed to mRNA-protein complexes, mRNPs. The mRNPs are then exported to the cytoplasm and the mRNAs are translated into proteins. The mRNAs containing in-frame premature stop codons are recognized and degraded in the nonsense-mediated mRNA decay process. This mRNA surveillence may also occur in the nucleus and presumably involves components of the translation machinery. Several translation factors have been detected in the nucleus, but their functional relationship to the dynamic protein composition of pre-mRNPs and mRNPs in the nucleus is still unclear.

Here, we have identified and characterized the translation initiation factor eIF4H in the dipteran Chironomus tentans. In the cytoplasm, Ct-eIF4H is associated with poly(A+) RNA in polysomes. We show that a minor fraction of Ct-eIF4H enters the nucleus. This fraction is independent on the level of transcription. CteIF4H could not be detected in gene-specific pre-mRNPs or mRNPs, nor in bulk mRNPs in the nucleus. Our immunoelectron microscopy data suggest that Ct-eIF4H associates with mRNP in the cytoplasmic perinuclear region, immediately as the mRNP exits from the nuclear pore complex.

Key words: Gene expression, Pre-mRNA processing, Translation


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the nucleus, pre-mRNAs – precursors to mRNA – associate with a large number of different proteins. These proteins are essential for intranuclear steps in gene expression, such as packaging, processing and export of pre-mRNAs. Many of these proteins – for example, hnRNP proteins (Dreyfuss et al., 1993Go; Krecic and Swansson, 1999Go; Dreyfuss et al., 2002Go) and splicing factors (Neubauer et al., 1998Go; Will and Lührmann, 2001Go) bind to the pre-mRNAs co-transcriptionally, resulting in the formation of pre-mRNA–protein complexes, pre-mRNPs. The composition of individual pre-mRNPs is dynamic. For example, splicing changes the protein composition of the pre-mRNP (Luo and Reed, 1999Go; Kataoka et al., 2000Go; Le Hir et al., 2000Go), and specific proteins are known to bind to pre-mRNP during transcription and to dissociate from the pre-mRNP before nucleocytoplasmic export through the nuclear pores complexes (NPCs) (Daneholt, 2001Go). Certain proteins that bind to pre-mRNA in the nucleus accompany the mRNA to the cytoplasm and determine the cytoplasmic fate of the mRNA (for a review, see Wilkinson and Shyu, 2001Go). For example, some proteins influence the cytoplasmic localization of mRNA (Hachet and Ephrussi, 2001Go; Johnstone and Lasko, 2001Go), and in oocytes, Y-box proteins bind to mRNA in the nucleus; these proteins are important during translation inactivation of the mRNAs in the cytoplasm (Matsumoto and Wolffe, 1998Go).

Spliced mRNAs associate with a protein complex, the exonexon junction complex (EJC). This complex, which contains REF/Aly, Y14, DEK, SRm160 and RNPS1, binds upstream of exon-exon junctions (Le Hir et al., 2000Go; Blencowe et al., 1998Go; McGarvey et al., 2000Go; Mayeda et al., 1999Go; Zhou et al., 2000Go; Kataoka et al., 2000Go; Kataoka et al., 2001Go; Kim et al., 2001Go; Lykke-Andersen et al., 2001Go). Some of the EJC components, as well as EJC-associated proteins such as Mago (Palacios, 2002Go) and the nonsense-mediated decay factor hUpf3 (Kim et al., 2001Go), accompany the mRNP to the cytoplasm. It has been suggested that such proteins are involved in discriminating between normal mRNAs and mRNAs containing in-frame stop codons, in a process called nonsense-mediated decay (NMD) surveillance (reviewed by Li and Wilkinson, 1998Go; Hentze and Kulozik, 1999Go; Hilleren and Parker, 1999Go; Kataoka et al., 2000Go; Lykke-Andersen et al., 2001Go; Maquat and Carmichael, 2001Go; Wilusz et al., 2001Go). NMD requires translation and can take place in the cytoplasm (Frischmeyer and Dietz, 1999Go; Zhang et al., 1997Go).

In the nucleus, the m7 G-cap-structure of the pre-mRNA binds the cap-binding complex, CBC, which contains the two cap-binding proteins CBP20 and CBP80. At least one of these, CBP20, binds co-transcriptionally (Visa et al., 1996aGo). The CBC stimulates pre-mRNA processing (Colot et al., 1996Go; Lewis et al., 1996Go; Fortes et al., 1999Go), but a direct role in nucleocytoplasmic mRNA export has not been established. In the cytoplasm, the CBC is replaced by the translation factor eIF4E at the cap structure, probably after the initial association with eIF4G (Fortes et al., 2000Go). The heterotrimeric complex between eIF4E, eIF4G and eIF4A, called eIF4F, together with eIF4B is believed to unfold the 5'-UTR (untranslated region) of the mRNA, an important step during the initiation of translation (Hershey and Merrick, 2000Go). The recently discovered eIF4H (Richter-Cook et al., 1998Go), is also involved at this stage of translation and has similar functions as eIF4B (Richter et al., 1999Go; Rogers et al., 1999Go; Rogers et al., 2001Go).

Recently, experimental data have been presented that suggest that translation takes place in the nucleus (Iborra et al., 2001Go; Brogna et al., 2002Go). A possible rationale for nuclear translation is that it is part of nuclear NMD (Dahlberg et al., 2003Go). If translation does occur in the nucleus, it has to be initiated, either by the known translation initiation factors or by a modified initiation process. It has been reported that the translation initiation factors eIF4E (Lejbkowicz et al., 1992Go; Lang et al., 1994Go; Dostie et al., 2000aGo), eIF4G (Etchinson and Etchinson, 1987Go; McKendrick et al., 2001Go) and eIF2 (Lobo et al., 1997Go) are present in the cell nucleus in significant amounts. Also, eIF4H has been reported to be present in the nucleus (Martindale et al., 2000Go). There are several possible explanations for the presence of these translation factors in the nucleus. One is that they are in fact involved in a nuclear translation process (Iborra et al., 2001Go; Brogna et al., 2002Go), possibly as part of nuclear NMD (Bühler et al., 2002Go). If so, there might be specific requirements for the initiation of nuclear translation (Fortes et al., 2000Go; Ishigaki et al., 2001Go). A second possibility is that they have so far unknown functions in pre-mRNA processing or export of mRNA. This possibility could be exemplified by eEF1A, which is present in the nucleus under certain conditions (Gangwani et al., 1998Go; Grosshans et al., 2000Go) and by eIF4E. In the case of eIF4E, this factor piggy-backs into the nucleus on 4E-T, a recently identified nucleocytoplasmic shuttling protein (Dostie et al., 2000bGo). eIF4E has been implicated in nuclear export of cyclin D1 mRNA (Rosenwald et al., 1995Go; Rousseau et al., 1996Go), a function that is dependent on cap binding (Cohen et al., 2001Go) and which is influenced by the promyelocytic leukemia protein PML (Topisirovic et al., 2002Go). A third possibility is that some translation initiation factors could bind to mRNAs already in the nucleus and accompany them to the cytoplasm, as a step in facilitating normal translation initiation. Finally, translation factors could simply be misplaced inside the nucleus without having a function there. It is important to investigate which translation factors are present in the nucleus and the functional relationship between these factors and synthesis, processing, transport and possible translation of mRNAs in the nucleus.

Here, we have isolated the gene encoding eIF4H from the dipteran Chironomus tentans, Ct-eIF4H. We have analyzed its cellular location and its relation to transcription and translation. We have especially used the experimental possibilities offered by the Balbiani ring (BR) genes and their large BR mRNP particles to test the hypothesis that Ct-eIF4H is bound to pre-mRNA/mRNA in the nucleus. Although we can show that CteIF4H is indeed present in the nucleus in small amounts, our data suggest that this translation initiation factor is not bound to pre-mRNA or mRNA in the nucleus. Instead, our data propose that Ct-eIF4H associates with mRNA in the cytoplasmic perinuclear region, immediately as the mRNA enters the cytoplasm.


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Biological material
Animals and cells
C. tentans was cultured as previously described (Meyer et al., 1983Go). A C. tentans embryonic epithelial cell line was cultured as described (Wyss, 1982Go). Defolliculated stage VI Xenopus laevis oocytes were prepared by collagenase treatment (Almouzni and Wolffe, 1993Go). Before microinjection, the oocytes were incubated over night at 18-19°C in OR2 medium containing 1 mM CaCl2 (Colman, 1984Go).

Antibodies
The coding part of the Ct-eIF4H gene was cloned into the pET-15b expression vector (Novagen) and expressed in Escherichia coli. His-tagged Ct-eIF4H was purified by Ni-NTA affinity chromatography (Qiagen) and used for immunization of both rabbits and hens. Anti-Ct-eIF4H polyclonal antibodies were affinity purified by chromatography on CNBr-activated Sepharose 4B (Amersham Biosciences AB) to which the protein had been coupled. Anti-hrp45 (Kiseleva et al., 1994Go), anti-hrp36 (Wurtz et al., 1996Go) and anti-hrp23 (Sun et al., 1998Go) monoclonal antibodies were a gift from B. Daneholt (Karolinska Institute, Stockholm, Sweden) and anti-CBP20 antibodies were a gift from I. W. Mattaj (EMBL, Heidelberg, Germany). The secondary antibodies used for immunocytology were: swine anti-rabbit Ig FITC (DAKO), diluted 1:100. The secondary antibodies used for western blots were: swine anti-rabbit Ig horse radish peroxidase (HRP), goat anti-mouse Ig HRP (DAKO) diluted 1:3000 and rabbit anti-chicken IgY HRP (SIGMA) diluted 1:20.000. Anti-rabbit immunoglobulin antibodies conjugated with 6 nm gold particles (Jackson ImmunoResearch Labs) were used as secondary antibodies for immunoelectron microscopy.

Cloning procedures
Total RNA was extracted from C. tentans salivary gland cells. In all, 400 salivary glands were dissected, fixed in 70% ethanol at 4°C and stored in glycerol:ethanol (1:1 by volume) at –20°C. RNA was extracted for 30 minutes at room temperature in 1 mM EDTA, 20 mM Tris-HCl pH 7.4, containing 0.5% SDS and 0.1 mg/ml proteinase-K, followed by phenol extraction and ethanol precipitation. Poly(A+) RNA was purified by binding to oligo(dT) cellulose (Poly(A) Quick, Stratagene). For cDNA synthesis, 5 µg of poly(A+) RNA was annealed with oligo(dT) at 70°C for 5 minutes. Reverse transcriptase (Superscript, BRL GIBCO) was added and synthesis was allowed to proceed for 1 hour at 42°C. Degenerate PCR primers, corresponding to the conserved RNP-1 and RNP-2 regions of the consensus-RBD present in many RNA-binding proteins, were used for amplification of corresponding regions in the cDNA, essentially as described (Kim and Baker, 1993Go). PCR products were analyzed by agarose gel electrophoresis. PCR products that were approximately 140 bp or 400 bp long were eluted from the agarose gel, cleaved with EcoRI and BamHI and cloned into a plasmid vector (Bluescript, Stratagene). cDNAs from individual clones were sequenced, and those exhibiting sequence homology to consensus-RBDs were selected. Full-length cDNA sequences were obtained by a combination of screening of a C. tentans tissue culture cDNA library, constructed in the lambda Zap vector (Stratagene), and direct isolation of fragments extending in the 5' or 3' direction (Marathon, Clontech). The genomic sequence organization was determined by analysis of several genomic PCR products, which together covered the entire gene. All exon-intron borders were sequenced and most introns were sequenced entirely. The DNA and protein sequences were analyzed by programs in the GCG package (Devereaux et al., 1984Go) and Biology Workbench package (San Diego Supercomputer Center, University of California, San Diego).

Protein preparation and western blotting
Nuclear and cytoplasmic extracts of C. tentans tissue culture cells were prepared essentially as described (Wurtz et al., 1996Go). To check for cytoplasmic contamination of the nuclear fraction, we used a cytoplasmic protein of unknown function. Proteins to be analyzed by western blotting were boiled in sample buffer (62.5 mM Tris-HCl pH 6.8, containing 10% glycerol, 2.3% SDS, 5% mercaptoethanol and 0.02% bromophenol blue) for 3 minutes and separated on 12% SDS-polyacrylamide gels. The separated proteins were transferred to PVDF-filters by semi-dry electrophoresis. HRP-labeled secondary antibodies were detected by the ECL method (Amersham Biosciences AB). Quantification of the bands was performed with a FUJIFILM LAS-1000 camera using the software Image Gauge V 3.45. Presented values are averages from three independent experiments.

Analysis of polysomes
C. tentans tissue culture cells were homogenized in 10 mM Tris-HCl pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 2 mM DTT and centrifuged at 20,000 g. The supernatant was centrifuged at 100,000 g in a AH-650 rotor (Sorvall) for 3 hours at 4°C to pellet polysomes. The pellet was washed and resuspended in 250 mM sucrose, 1 mM DTT, 0.1 mM EDTA, 20 mM Tris-HCl pH 7.5. KCl was added to a final concentration of 0.5 M and ribosomes were repelleted at 100,000 g for 4 hours at 4°C. The supernatant and the pellet was analyzed by western blotting.

Immunocytological localization
Cultured C. tentans diploid cells were prepared and stained with anti-Ct-eIF4H antibodies essentially as described (Baurén et al., 1996Go). Chromosomes were isolated from salivary glands of C. tentans fourth instar larvae and probed with anti-Ct-eIF4H antibodies, essentially as described (Kiseleva et al., 1994Go). Anti-hrp45 antibodies were used for parallel preparations. These antibodies stain hrp45 bound to nascent BR pre-mRNPs (Baurén et al., 1996Go) and served as a positive control (data not shown). The secondary antibody alone and pre-immune serum did not give any significant labeling (data not shown). All preparations were mounted in antifade medium (VectaShield, Vector Laboratories) and viewed and photographed in an Axioplan II microscope (Zeiss).

Immunoelectron microscopy
Immunoelectron microscopy of ultrathin cryosections of C. tentans salivary gland cells was performed according to Tokuyasu (Tokuyasu, 1980Go) as described (Visa et al., 1996bGo). The specimens were examined and photographed in a Zeiss CEM 902 electron microscope at 80 kV. Negative controls with only secondary antibodies and positive controls with anti-hrp45 antibodies were performed in parallel (data not shown). For quantification of Ct-eIF4H labeling, all gold particles present in 500 nm wide regions on both sides of the nuclear membrane were counted. In a 150 nm cytoplasmic perinuclear region, the location of all gold particles were analyzed and it was determined if they were associated with NPCs or located between NPCs. In the digitized micrographs, the gold particles were enlarged to improve visualization.

Expression of Ct-eIF4H in Xenopus oocytes and analysis of its cellular location
The coding part of Ct-eIF4H was cloned into the plasmid vector pßGFP/RN3P (Zernika-Goetz et al., 1996Go). Capped RNA was transcribed in vitro (Ambion) and injected into the cytoplasm of Xenopus oocytes, using a Nanoliter 2000TM (World precision instruments) as described before (Belikov et al., 2000Go). After 1-2 days, cytoplasm and nuclei were manually dissected and the presence of Cte-IF4H was detected in western blots using anti-Ct-eIF4H antibodies. The antibodies detected a few endogenous Xenopus proteins in both injected and uninjected cells. In certain oocytes, the nuclear membrane was manually removed from isolated nuclei and the nuclear membrane and the nucleoplasm were analyzed separately. To assay for the influence of increased transcription, Ct-eIF4H mRNA was co-injected with in vitro transcribed mRNA for the glucocorticoid receptor and a plasmid containing the herpes simplex thymidine kinase gene (HS VTK), driven by the MMTV promoter. Nuclei and cytoplasm were analyzed in oocytes treated or not treated with 1 µM of the synthetic glucocorticoid triamcinolone acetonide (TA) for 8 hours to induce transcription from the MMTV promoter. As a reference for the amounts of material loaded in each lane in the western blots, the blots were probed with an anti-BRG-1 antibody, thus detecting endogenous BRG-1 (Östlund-Farrants et al., 1997Go). The intensity of the bands were quantified as explained above.

Analysis of poly(A+) RNA
C. tentans tissue culture cells were UV-irradiated and poly(A+) RNA was isolated from cytoplasm using Oligotex according to the manufacturer (Qiagen), and from nuclei essentially as described (Pinol-Roma et al., 1989Go). After elution of the RNA from the oligo(dT) cellulose and RNase treatment, the co-purified proteins were analyzed by western blotting.

Immunoprecipitation of pre-mRNP and mRNP complexes
Pre-mRNP and mRNP complexes were immunoprecipitated from nuclear extract of C. tentans tissue culture cells essentially as described (Sun et al., 1998Go). In brief, monoclonal anti-hrp23 antibody or polyclonal anti-Ct-eIF4H antibodies were added to nuclear extracts in the presence of 0.1% NP-40, and incubated for 90 minutes at 4°C with gentle rotation. Rabbit anti-mouse immunoglobulins (DAKO), covalently coupled to protein G-Sepharose beads (Zymed Laboratories) or protein G-Sepharose beads were added and the rotation was continued for 90 minutes at 4°C. The sepharose beads were washed three times with PBS containing 0.1% NP-40 and once with PBS. The immunoprecipitated complexes were eluted with 0.5% SDS at room temperature. The proteins were precipitated with acetone and subsequently analyzed by western blotting.


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Cloning and characterization of Ct-eIF4H
In a screen for proteins that are associated with mRNAs, we took advantage of the fact that such proteins often contain a conserved RNA-binding domain, RBD (Varani and Nagai, 1998Go). Messenger RNA was purified from C. tentans salivary gland cells and cDNA was obtained by using oligo (dT) primers. Two different degenerate primers, corresponding to the conserved RNP-1 and RNP-2 amino acid sequences present in the RBD in RNA-binding proteins, were used for PCR. The PCR fragments were cloned into a plasmid vector and individual cDNAs were sequenced. One of the gene fragments that coded for part of an RBD was used to obtain the entire coding region of the corresponding gene from a C. tentans cDNA library. The 1460 bp long cDNA sequence encoded a protein consisting of 316 amino acids. On the basis of sequence similarities and properties described below, we concluded that this protein is C. tentans eIF4H (Ct-eIF4H).

In Fig. 1, Ct-eIF4H is compared to its closest sequence homologues in Drosophila melanogaster (41% identity) and Homo sapiens (32% identity). In Fig. 1A it is evident that eIF4H in the three species have a very similar RBD in the N-terminus and also a highly conserved C-terminus. The greatest difference between the proteins is found in the central region. Here, the proteins differ in length and sequence. The human eIF4H is considerably shorter than eIF4H in dipterans, and the size difference is localized to the central region. Two splice variants of the human eIF4H have been detected that differ by 20 amino acid residues, and this splice variation is present in the central region. Also, the dipteran proteins in the central region are different in each of the species mentioned. In all three species, the central region is rich in glycine residues and Ct-eIF4H contains six RGG motifs. Such motifs can contribute to RNA binding (Kiledjian and Dreyfuss, 1992Go) and protein-protein interactions (Gary and Clarke, 1998Go).



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Fig. 1. Sequence variation in eIF4H between species. (A) Comparison of amino acid sequences for eIF4H in H. sapiens (accession number Q15056), D. melanogaster (accession number CG4429) and C. tentans (accession number AJ511864). The N-terminus, with its RNA-binding domain, and the C-terminus are similar in the three species. The largest variation is in the central part of the protein, where all proteins are rich in glycine residues. Conserved residues are highlighted: black indicates identical residues; grey indicates related residues. (B). The intron positions (indicated by the arrows) in eIF4H in H. sapiens, D. melanogaster and C. tentans are shown. Gaps (larger than two amino acid residues) have been introduced in the proteins in accordance with the alignment in Fig. 1A.

 

By in situ hybridization, the Ct-eIF4H gene was located to chromosome I, region 16C (data not shown). The exon-intron structure of the Ct-eIF4H gene was deduced by comparing genomic and cDNA PCR fragments. Five introns were identified and the exon-intron borders were sequenced. The introns are between 67 bp and 233 bp long, and their positions are indicated in Fig. 1B. The eIF4H gene in D. melanogaster contains four introns. The introns 1, 2, 3 and 4/5 are in very similar positions in C. tentans and D. melanogaster. The intron corresponding to intron 4 in the Ct-eIF4H gene is lacking in the D. melanogaster gene. In the human gene, there are six introns. Only intron 6 is in the same position as the most 3' located intron in the dipteran genes. Exon 5 in humans is known to be subject to exon skipping/inclusion to produce two splice variants.

Ct-eIF4H is present in the cytoplasm and bound to mRNA in polysomes
Polyclonal antibodies were raised in rabbits and in hens against the recombinant Ct-eIF4H. In both cases the antibodies specifically detected one single protein with the relative mobility of 36 kDa in western blots of C. tentans cell extracts (Fig. 2A). When cytoplasm and nuclei in C. tentans tissue culture cells were biochemically separated, approximately 95% of Ct-eIF4H were detected in the cytoplasmic fraction and approximately 5% in the nuclear fraction (Fig. 2A, lanes 1 and 2). In agreement, immunofluorescence localization of Ct-eIF4H in C. tentans tissue culture cells showed that Ct-eIF4H is present throughout the cytoplasm (Fig. 2B).



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Fig. 2. Localization of Ct-eIF4H. (A) Western blot analysis of cytoplasmic and nuclear fractions of C. tentans diploid cells. The cells were biochemically fractionated into a cytoplasmic (C) and a nuclear (N) fraction. The fractions were analyzed by western blotting, using anti-Ct-eIF4H antibodies. The majority (about 95%) of Ct-eIF4H is present in the cytoplasmic fraction, but a small amount (about 5%) is found in the nuclear fraction. The extra band in the cytoplasmic fraction (approximately 3% of the signal in this lane) was specific for the antibody, but we do not know the identity of this band. In lanes 3 and 4, the fractions were probed with an antibody specific for a cytoplasmic protein to check for contamination of cytoplasm in the nuclear extract. (B) C. tentans diploid cells were stained with anti-Ct-eIF4H antibodies and detected by immunofluorescence (left panel). A predominant cytoplasmic staining was seen. Consistently, a weak granular staining was detected in the nucleus. DNA, in the same cell, stained with DAPI (right panel). Bar, 1 µm.

 

Translation factors belonging to the eIF4 group are co-purified with polysomes in a salt-dependent manner (Merrick, 1979Go). We isolated polysomes from C. tentans tissue culture cells. In Fig. 3A (lane 1), we showed that Ct-eIF4H co-sediments with polysomes. We also showed that when isolated polysomes were treated with 0.5 M KCl, Ct-eIF4H dissociated from the polysomes (Fig. 3A, lanes 2 and 3). To further analyze the association of Ct-eIF4H with polysomes, we UV-irradiated C. tentans tissue culture cells and purified cytoplasmic poly(A+) RNA. We then found that Ct-eIF4H was co-purified with the poly(A+) RNA and that this co-purification was dependent on UV-irradiation (Fig. 3B). The hrp36 protein, known to be bound to mRNA in the cytoplasm, served as a positive control (Fig. 3B). The Ct-RBD-1 protein, known to be present in the cytoplasm and partly associated with 40S ribosomal subunits served as a negative control (data not shown). In summary, Ct-eIF4H is present throughout the cytoplasm, and is probably associated with poly(A+) RNA in polysomes.



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Fig. 3. Ct-eIF4H is present in polysomes and can be UV-cross-linked to cytoplasmic poly(A+) RNA. (A) Polysomes from C. tentans tissue culture cells were pelleted by centrifugation. Ct-eIF4H, detected by western blotting, was present in the pelleted polysomes (lane 1). The polysomes were resuspended, treated with 0.5 M KCl and repelleted. Ct-eIF4H was then released from the polysomes (lane 2) and found in the supernatant (lane 3). (B) C. tentans tissue culture cells were UV-irradiated, and poly(A+) RNA was isolated from cytoplasmic extracts. After elution of the RNA from the oligo (dT) cellulose and RNase treatment, the co-purified proteins were analyzed by western blotting. Ct-eIF4H (lane 1) and hrp36 (lane 3) co-purifies with poly(A+) RNA after UV-cross-linking but not when UV-cross-linking was left out (lanes 2 and 4, respectively).

 

Ct-eIF4H is present in the nucleus, but not associated with pre-mRNA or mRNA
In the cellular fractionation experiments (Fig. 2A, lane 2) and in the immunostaining of the tissue culture cells (Fig. 2B, left panel), we detected Ct-eIF4H not only in the cytoplasm, but also to a minor extent in the nucleus. In the biochemical fractionations, we detected no contamination from the cytoplasm in the nuclear fraction, using a control antibody specific for a cytoplasmic protein (Fig. 2A, lanes 3 and 4). The immunofluorescence staining of the nuclei, although weak, was significant compared with controls and had a granular pattern throughout the nucleus.

To further analyze the nuclear localization of Ct-eIF4H, we used Xenopus oocytes, C. tentans salivary gland cells and C. tentans diploid cells. Xenopus oocytes allow experimental expression of Ct-eIF4H, combined with efficient isolation of nuclei, and analyses of the relation between the level of transcription and nuclear content of Ct-eIF4H. Ct-eIF4H was expressed in Xenopus oocytes by the injection of in vitro transcribed RNA. Subsequently, we analyzed the cellular location of the protein. Fig. 4A (lanes 1-4) shows that Ct-eIF4H was specifically detected both in the cytoplasm and in the nucleus. It also shows that Ct-eIF4H was present inside the nucleus and not solely bound to the outside of the nuclear membrane (Fig. 4A, lanes 5 and 6). To address whether the nuclear content of Ct-eIF4H is influenced by transcription activity we took advantage of the glucocorticoid hormone inducible MMTV promoter. Hormone induction of this promoter after injection into Xenopus oocytes results in a strong induction of transcriptional activity and a concomitant chromatin remodeling of all injected copies, while the uninduced promoter remains virtually silent (Belikov et al., 2000Go). By injecting 10 ng of the 10.25 kb construct pMTV:M13 (Belikov et al., 2000Go) as ssDNA we achieved about 1-2x109 copies of the gene in each oocyte. The hormone induction resulted in a massive increase in MMTV promoter-driven transcription (data not shown). However, this did not result in any significant change in the distribution of Ct-eIF4H, which was coexpressed by RNA injection into these oocytes (Fig. 4B).



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Fig. 4. Analysis of the localization of Ct-eIF4H in Xenopus oocytes. (A) Nuclei (N) and cytoplasm (C) were dissected from injected and uninjected Xenopus oocytes. The presence of Ct-eIF4H was analyzed by western blotting (lanes 1-4). The nuclei from injected oocytes were further dissected and the nucleoplasm (lane 5) and the nuclear membrane (lane 6) were analyzed separately. The position of Ct-eIF4H is indicated. The additional bands represent endogenous Xenopus proteins that were present in both uninjected and injected oocytes. (B) Hormone-induced transcription of the HS VTK gene did not increase the nuclear content of Ct-eIF4H. Oocytes, injected with Ct-eIF4H mRNA and the HS VTK gene construct, were dissected and the content of Ct-eIF4H in nuclei (N) and cytoplasm (C) was analyzed by western blotting in the absence (lanes 3 and 4) or in the presence (lanes 5 and 6) of hormone. Uninjected oocytes were analyzed as a control (lanes 1 and 2). As an internal control for the amount of material loaded in each lane, the endogenous BRG-1 protein on the same filter was detected with a specific antibody. Quantification of the signals showed that the amount of material loaded in lane 5 was approximately 1.3 times the amount loaded in lane 3, which explains the apparent difference in strength of the Ct-eIF4H band in these two lanes.

 

C. tentans salivary gland cells allow a detailed analysis of the nuclear distribution of Ct-eIF4H in relation to specific gene loci and gene-specific mRNA-protein complexes (mRNPs). We stained isolated polytene chromosomes from C. tentans salivary gland cells with the anti-Ct-eIF4H antibodies. As seen in Fig. 5, we could not detect any significant staining of the transcriptionally highly active Balbiani ring (BR) gene loci. Furthermore, we could not detect any staining of other gene loci on the polytene chromosomes. This finding strongly suggests that Ct-eIF4H is not associated with nascent pre-mRNAs at the gene loci.



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Fig. 5. Ct-eIF4H is not bound to nascent pre-mRNAs. (A) Isolated C. tentans polytene chromosome IV was probed with anti-Ct-eIF4H antibodies. No staining of the transcriptionally active BR gene loci (BR1, BR2 and BR3), or of other gene loci could be detected. Bar, 10 µm. (B) The same chromosome viewed in phase contrast.

 

To investigate whether Ct-eIF4H binds to mRNPs in the interchromatin after release of the processed mRNPs from the gene loci, we used immunoelectron microscopy. In C. tentans salivary gland cells, transcripts from the BR1 and BR2 genes form 7 nm thick RNA-protein fibers which fold into mRNP particles with a diameter of 50 nm (Lönnroth et al., 1992Go). These mRNP particles are morphologically distinguishable as they are synthesized on the gene and as they subsequently are transported through the interchromatin. In the electron microscope, we could not detect any Ct-eIF4H labeling of the nascent BR pre-mRNP, in agreement with the lack of immunostaining of isolated chromosomes. We detected specific gold particles, indirectly labeling Ct-eIF4H, throughout the interchromatin, but no labeling of BR mRNP particles could be detected (Fig. 8A).



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Fig. 8. Electron micrographs of anti-Ct-eIF4H antibody labeling in the interchromatin and in the cytoplasmic perinuclear region in C. tentans salivary gland cells. A schematic drawing of each micrograph is shown to the right to simplify interpretation. The nuclear membrane is represented by the dashed lines and the gaps indicate the positions of NPCs as shown in the schematic drawing of B. In each micrograph, the nucleus (Nuc) and cytoplasm (Cyt) are indicated. Gold particles are denoted by black dots. (A) BR mRNP particles (circles marked BR in the schematic drawing) in the interchromatin and docking at NPCs (arrow) were not labeled by the anti-Ct-eIF4H antibodies. (B) A Ct-eIF4H-specific gold particle associated with the cytoplasmic side of a NPC. This is also seen for the two gold particles in the cytoplasmic perinuclear region in A. (C,D) Ct-eIF4H-specific gold particles associated with electron-dense fibers extending from NPCs into the cytoplasmic perinuclear region. These fibers represent extended BR mRNP particles during passage through the NPCs. In the schematic drawing these fibers are marked BR. Bar, 200 nm.

 

To further analyze whether Ct-eIF4H is associated with mRNAs in general in the nucleus, we isolated poly(A+) RNA from nuclear preparations of C. tentans diploid tissue culture cells after UV-irradiation. We could not detect Ct-eIF4H UV-cross-linked to nuclear poly(A+) RNA (Fig. 6). The hnRNP protein hrp36 served as a positive control and could be detected. Even though hrp36 is likely to be bound in several copies to each mRNA, a comparison of Fig. 6 and Fig. 3B shows that relative to hrp36, Ct-eIF4H associated to poly(A+) RNA can be detected after UV-cross-linking in the cytoplasm, but in the nucleus it is below the level of detection.



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Fig. 6. Ct-eIF4H is not bound to nuclear poly(A+) RNA. C. tentans tissue culture cells were or were not UV-irradiated and poly(A+) RNA was isolated from nuclear extracts. The co-purified proteins were analyzed by western blotting. Ct-eIF4H did not co-purify with poly(A+) RNA after UV-cross-linking (lanes 1 and 2), whereas hrp36 did (lanes 3 and 4).

 

We also addressed whether the nuclear Ct-eIF4H could be detected in pre-mRNP or mRNP complexes in C. tentans diploid cells. It has previously been shown that the SR protein, hrp45 (Alzhanova-Ericsson et al., 1996Go) and the SR related protein hrp23 (Sun et al., 1998Go) (Björk et al., in preparation) are restricted to the nucleus and that they are both present in pre-mRNP and mRNP complexes. We used anti-hrp23 antibodies in immunoprecipitations of nuclear extracts. We then asked whether we could detect Ct-eIF4H in the precipitated complexes. We found that Ct-eIF4H could not be co-immunoprecipitated with hrp23 (Fig. 7B). By contrast, we could co-immunoprecipitate hrp45 and CBP20 (Fig. 7B). We also used anti-Ct-eIF4H antibodies for immunoprecipitation. In this case we could only precipitate Ct-eIF4H, but not hrp23, hrp45 or CBP20 (Fig. 7A). These results suggest that Ct-eIF4H is not generally present in pre-mRNP or mRNP complexes in the nucleus.



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Fig. 7. Ct-eIF4H cannot be immunoprecipitated together with proteins present in pre-mRNP and mRNP complexes in the nucleus. Nuclear extracts (NE) from C. tentans tissue culture cells were immunoprecipitated with antibodies directed against either Ct-eIF4H (A) or the nuclear mRNP protein hrp23 (B). Immunoprecipitated (IP) complexes were analyzed by western blotting. Anti-Ct-eIF4H antibodies precipitated Ct-eIF4H, but not hrp23 and hrp45 (both are known to be associated with pre-mRNPs in the nucleus) or CBP20. Anti-hrp23 antibodies precipitated hrp23, hrp45 and CBP20 but not Ct-eIF4H.

 

Ct-eIF4H is present in the cytoplasmic perinuclear region, preferentially close to nuclear pore complexes
If Ct-eIF4H does not bind to mRNA in the nucleus, the question is where in the cell this association takes place. To attempt to answer this question, we used immunoelectron microscopy to study the location of Ct-eIF4H in the cytoplasm and in the interchromatin, especially in relation to the NPC in the nuclear membrane and to the export of BR mRNP. We detected Ct-eIF4H throughout the cytoplasm in close connection to the rough endoplasmic reticulum (data not shown), in agreement with our immunofluorescence data (Fig. 2B). The labeling was evenly distributed in the cytoplasm all the way up to the outer nuclear membrane. The number of gold particles specific for Ct-eIF4H was approximately the same in a 500 nm region closest to the outer nuclear membrane compared to the proceeding 500 nm cytoplasmic region. In the nucleus, specific immunolabeling was observed throughout the entire interchromatin. We detected no labeling of the chromatin (data not shown), in agreement with our imunofluorescence data (Fig. 5). To compare the distribution of specific gold particles on both sides of the nuclear membrane, we counted all the gold particles also in two 500 nm regions on the nuclear side of the membrane. The number of gold particles in the nuclear 500 nm region closest to the inner nuclear membrane was about ten times lower than on the corresponding cytoplasmic region (Table 1). The same was true for the proceeding 500 nm nuclear region. Even though the interchromatin was specifically labeled, we could not detect any labeling of BR mRNP particles (Skoglund et al., 1986Go) in the interchromatin, close to the nuclear membrane or docked at the basket structure of the NPCs (Fig. 8A).


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Table 1. Distribution of Ct-eIF4H-specific gold particles on the nuclear and cytoplasmic sides of the nuclear membrane

 

In the cytoplasm, we focused on the Ct-eIF4H-specific labeling in the cytoplasmic perinuclear region, defined as a 150 nm region closest to the nuclear membrane. Here, labeling was about as frequent as within the proceeding 350 nm from the nuclear membrane (Table 1). When we analyzed the distribution along the nuclear membrane, we found that almost 80% of the gold particles in the 150 nm perinuclear region were associated with NPCs (Fig. 8A,B), whereas about 20% of the gold particles were located between the NPCs. This suggests that the majority of Ct-eIF4H present in the cytoplasmic perinuclear region is not bound to mRNAs associated with the polysomes that are bound to the outer nuclear membrane.

Occasionally, a morphologically identifiable BR mRNP particle can be seen exiting an NPC at the cytoplasmic side as an extended mRNP fiber. Such mRNP fibers exit with their 5' end first. The fibers have been morphologically described as being connected to particles, which have the dimensions of ribosomes (Mehlin et al., 1992Go). We detected such electron-dense fibers extending from the NPCs and associated with Ct-eIF4H-specific gold particles (Fig. 8C,D). This suggests that Ct-eIF4H becomes bound to BR mRNP fibers immediately as these are exposed in the cytoplasm during translocation through the NPCs.

In summary, our immunoelectron microscopy data show that Ct-eIF4H is not bound to BR mRNP particles as these are docking at the nuclear side of the NPC. In the cytoplasmic perinuclear region, Ct-eIF4H is preferentially located close to the NPCs. Our data suggest that Ct-eIF4H associates with BR mRNP particles as these exit the NPCs, even before the entire mRNP has been translocated, and then accompanies the BR mRNP to the endoplasmic reticulum.


    Discussion
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 Summary
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 Materials and Methods
 Results
 Discussion
 References
 
It is important to analyze when and where mRNA interacts with the translation machinery. Translation and most probably surveillance of mRNA translatability are dependent on correct association between the mRNA and the 40S ribosomal subunit. Therefore, special attention should be given to factors that are involved in the initiation of translation.

The eIF4B/eIF4H proteins
We have studied the translation initiation factor eIF4H in C. tentans. This translation factor has been discovered only recently (Richter-Cook et al., 1998Go). It is apparent from the sequence alignment in Fig. 1 that there is substantial sequence variation in eIF4H between species. In particular, a central region, rich in glycine, arginine and asparagine residues, is variable, both as to sequence and to length. In mammals, a part of the central region interacts with a herpes simplex virus nuclease as part of viral-induced degradation of mRNAs that undergo cap-dependent translation (Feng et al., 2001Go; Everly et al., 2002Go). The N-terminus, which contains the RBD, and the C-terminus are conserved to a high extent. This is also true for eIF4H sequence homologues in Anopheles gambiae and Mus musculus. The D. melanogaster genome encodes an additional eIF4H sequence homologue. This protein is longer and has less sequence identity compared with human eIF4H. However, it is more similar to eIF4H than to eIF4B.

We noted that the RBD and the C-terminus in eIF4H are relatively well conserved compared with corresponding regions in mammalian eIF4B. Like eIF4B, eIF4H stimulates the ATPase and helicase activities of eIF4A (Richter et al., 1999Go; Rogers et al., 2001Go). In these and other assays, eIF4B and eIF4H exhibit an additive stimulatory behaviour, and it is possible that the two proteins can complement each other (Rogers et al., 2001Go). In mammals, eIF4B is considerably longer than eIF4H. It extends approximately 60 amino acid residues at the N-terminus and 230 residues at the C-terminus. As for Ct-eIF4H, eIF4B has an extra region, approximately 70 amino acid residues long, after the RBD, compared with mammalian eIF4H. This region of the eIF4B is part of the DRYG domain (Milburn et al., 1990Go; Naranda et al., 1994Go), which is rich in aspartic acid, arginine, tyrosine and glycine and is responsible for dimerization of eIF4B (Méthot et al., 1996Go). There is no clear sequence similarity between the DRYG domain and the glycine-rich central region in dipteran eIF4H.

In both Saccharomyces cerevisiae (Tif3p) and Schizosaccharomyces pombe (Sce3p), there is an eIF4B homologue, but no eIF4H homologue has been detected. If only one eIF4B/eIF4H protein is present in unicellular eukaryotes and a variable number is present in D. melanogaster and mammals, including pre-mRNA processing variants, it is possible that evolution of eIF4H/eIF4B has allowed mRNA-specific variation in translation initiation. Tissue-specific differences in mRNA levels for eIF4H and eIF4B have been found, as well as functional variations in biochemical assays (Richter et al., 1999Go).

Translation initiation factors in the nucleus
Several translation initiation factors are present not only in the cytoplasm but also in the cell nucleus, including the eIF4 factors, eIF4E (Lejbkowicz et al., 1992Go; Lang et al., 1994Go; Dostie et al., 2000aGo) and eIF4G (Etchinson and Etchinson, 1987Go; McKendrick et al., 2001Go). Our results show that CteIF4H is present in the nucleus in diptera and Xenopus. It has also been reported that eIF4H can be present in mammalian nuclei (Martindale et al., 2000Go). The function of these translation initiation factors in the nucleus is unclear. eIF4E is imported to the nucleus (Dostie et al., 2000bGo) where it is concentrated in nuclear bodies, often associated with PML bodies (Cohen et al., 2001Go; Topisirovic et al., 2002Go; Strudwick and Borden, 2002Go). eIF4E is bound to at least some mRNAs in the nucleus, presumably to the mRNA cap (Dostie et al., 2000aGo; Lejeune et al., 2002Go). These mRNAs do not contain introns and are not bound by the EJC or by the NMD factors Upf2, Upf3 and Upf3X (Lejeune et al., 2002Go; Ishigaki et al., 2001Go). For these reasons, eIF4E-bound nuclear mRNAs are probably not the substrate for nuclear NMD. It has been suggested that eIF4E is involved in exporting a subset of mRNAs, including cyclin D1 mRNA (Rosenwald et al., 1995Go; Rousseau et al., 1996Go). This function is dependent on cap binding and influenced by the PML protein, which reduces the affinity of eIF4E for the mRNA cap (Cohen et al., 2001Go; Kentsis et al., 2001Go). eIF4G interacts with the nuclear mRNA CBC and colocalizes with splicing factors in speckles. In vitro, the protein is associated with spliceosomes, but it does not appear to participate directly in splicing (McKendrick et al., 2001Go). The CBC is known to influence pre-mRNA processing (Lewis et al., 1996Go; Flaherty et al., 1997Go) and mRNA export (Jarmolowski et al., 1994Go; Visa et al., 1996aGo) and to facilitate CBC replacement by eIF4F (Fortes et al., 1999Go; McKendrick et al., 2001Go). Because some translation initiation factors are associated with the CBC, this raises the possibility that the factors could influence any of these processes.

Several other translation components have also been located in the nucleus (Brogna et al., 2002Go; Pederson and Politz, 2000Go; Wilkinson and Shyu, 2002Go), and it has been reported that translation takes place inside the nucleus (Iborra et al., 2001Go; Brogna et al., 2002Go). Others have found that efficient nuclear export occurs, in that several translation factors are present in the nucleus in very small amounts (Bohnsack et al., 2002Go; Calado et al., 2002Go), making it unlikely that nuclear translation takes place at any significant level. The functional significance of possible nuclear translation is unclear, but it may be connected to the mRNA quality assurance mechanism, NMD (Maquat and Carmichael, 2001Go; Maquat, 2002Go). Messenger RNAs are thought to be subjected to one initial round of translation, to ensure that they do not contain premature in-frame nonsense codons. This process is thought to occur before the mRNAs are released into the cytoplasm (Hentze and Kulozik, 1999Go; Maquat and Carmichael, 2001Go; Bühler et al., 2002Go), but conclusive experimental data are not yet available to rule out alternative possibilities (Dahlberg et al., 2003Go).

Ct-eIF4H binds to BR mRNA in the cytoplasmic perinuclear region
We have shown that Ct-eIF4H is present in small amounts in the cell nucleus. We have, in four different types of experiments – UV-cross-linking to poly(A+) RNA (Fig. 6), co-immunoprecipitation (Fig. 7), immunocytological staining of isolated chromosomes (Fig. 5) and immunoelectron microscopy (Fig. 8) – failed to detect an association between eIF4H and pre-mRNA/mRNA. We therefore suggest that Ct-eIF4H is not bound to gene-specific pre-mRNPs or mRNPs, nor to nuclear mRNPs in general. By contrast, our data show that Ct-eIF4H is bound to mRNAs in the cytoplasm, and our biochemical and immunoelectron microscopy data suggest that these mRNAs are present in ER-bound polysomes.

By studying the BR mRNP particles, which allow analysis at the level of individual mRNPs, we showed that Ct-eIF4H does not bind to these gene-specific mRNPs in the nucleus. The most plausible explanation for Ct-eIF4H not binding to mRNA in the nucleus is that other factors necessary for binding are not present or that some factors prevent binding. It is known that the BR mRNP particle changes morphology on translocation through the NPC and that some proteins leave the mRNP at this stage (Daneholt, 2001Go). In our immunoelectron microscopy study, we could not detect Ct-eIF4H binding to BR mRNP particles on the nuclear side of the NPCs. However, our data show that Ct-eIF4H is present in close association with the NPCs at their cytoplasmic side as the 5' end of the BR mRNP emerges from the NPC. Our data furthermore suggest that Ct-eIF4H becomes associated with the BR mRNP very close to the NPC, probably before the entire mRNP has been translocated through the NPC.

Initiation of translation and NMD
We are unaware of any studies showing that eIF4B, the functional partner to eIF4H, is present in the nucleus. Nor has it been reported that eIF4A, a subunit of eIF4F, is present in the nucleus. Several reports suggest that the initial round of translation occurs via a mechanism that may involve a different initiation complex compared with the subsequent steady-state initiation complex. The initial round of translation may be part of NMD and take place before CBP80-CBP20 are exchanged at the mRNA cap structure (Fortes et al., 2000Go; Ishigaki et al., 2001Go; Lejeune et al., 2002Go). eIF4F is normally engaged in the binding of the mRNA to the 40S ribosomal subunit. It is therefore possible that there are distinctly different translation initiation complexes in the cell. Perhaps only the steady-state initiation complex in the cytoplasm includes all eIF4 factors, thereby ensuring highly efficient translation initiation.

We have investigated the association between BR mRNP and Ct-eIF4H. The BR mRNP is unfolded when it is translocated through the NPC with its 5' end first. It is not refolded on the cytoplasmic side of the NPC (Mehlin et al., 1992Go). It is known that newly synthesized BR mRNA rapidly form polysomes (Daneholt et al., 1977Go) and becomes associated with the endoplasmic reticulum (Lönn, 1977Go). It is therefore possible that translation of BR mRNA is normally initiated in the cytoplasmic perinuclear region and that the observed association between Ct-eIF4H and BR mRNP reflects such initiation events. If Ct-eIF4H is involved in the initiation of translation coupled to NMD surveillance of the BR mRNAs, our data suggest that NMD does not take place inside the nucleus. Instead, it could occur in close connection to nucleocytoplasmic export, in the cytoplasmic perinuclear region. This is one possibility that could explain association of NMD with the nucleus (Dahlberg et al., 2003Go). To obtain a more complete picture, we need to further analyze the spatial association of other translation initiation factors with mRNPs. This requires highly specific antibodies to these factors.


    Acknowledgments
 
We wish to thank Kerstin Bernholm and Inger Granell for excellent technical assistance and Dr I. W. Mattaj for the anti-CBP20 antibodies and Dr B. Daneholt for the anti-hrp45, anti-hrp36 and anti-hrp23 antibodies. We also thank Dr Neus Visa and Birgitta Björkroth for valuable advice. This work was supported by the Swedish Research Council (Natural and Engineering Sciences) and the Magnus Bergvalls Stiftelse Swedish Cancer Foundation.


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 Top
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 Introduction
 Materials and Methods
 Results
 Discussion
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