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First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00766

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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

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

View larger version (54K): [in a new window]   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.

View larger version (30K): [in a new window]   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.

View larger version (20K): [in a new window]   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).

View larger version (30K): [in a new window]   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.

View larger version (28K): [in a new window]   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.

View larger version (56K): [in a new window]   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.

View larger version (19K): [in a new window]   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).

View larger version (39K): [in a new window]   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.