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First published online March 12, 2004
doi: 10.1242/10.1242/jcs.00992

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CRM1 and Ran are present but a NES-CRM1-RanGTP complex is not required in Balbiani ring mRNP particles from the gene to the cytoplasm

Jian Zhao^*, Shao-Bo Jin and Lars Wieslander

Department of Molecular Biology and Functional Genomics, Stockholm University, SE-106 91 Stockholm, Sweden

View larger version (121K): [in a new window]   Fig. 4. Immunoelectron microscopy localisation of Ct-CRM1 in salivary gland cell nuclei. (A) View of a region of an active BR gene locus. Short stretches of many different transcribing BR genes and of different parts of the BR gene are seen. Nascent BR pre-mRNP particles are labelled by the secondary, gold-conjugated antibody. The gold particles are approximately 6 nm in diameter. (B) A complete active BR gene is shown schematically with the growing BR pre-mRNP particles. At the proximal (p) part of the gene, the BR pre-mRNPs form short fibres. These fold into larger and larger spherical pre-mRNP particles seen in the middle (m) and distal (d) parts of the active gene. (C-F) sections highlight areas containing essentially proximal, middle and distal parts of the gene, respectively (the sizes of the nascent mRNPs are different according to the drawing in B.) Gold particles can be seen labelling the BR pre-mRNP in all three cases (arrows). Bars, 200 nm.

View larger version (28K): [in a new window]   Fig. 1. Specificity of the anti-CRM1 and anti-Ran monoclonal antibodies. (A) Western blot of extracts from C. tentans tissue culture cells and salivary gland cells, probed with the anti-CRM1 antibody. (B) Western blot analysis of the expression of the N-terminal part of Ct-CRM1 (residues 1-329) in E. coli. Lane 1, before isopropyl ß-D-thiogalactopyranoside (IPTG) induction of expression. In lane 2, a polypeptide migrating at approximately 36 kDa (arrow) was recorded after induction by IPTG. (C) Western blot of protein extracts from C. tentans tissue culture cells (lane 1) and salivary gland cells (lane 2), probed with the anti-Ran antibody. (D) Expression of a GST-Ct-Ran fusion protein in E. coli. In lane 1, no protein was seen before induction with IPTG. In lane 2, the fusion protein (arrow) was detected as well as smaller polypeptides, specifically reacting with the anti-Ran antibody. In A-D, M indicates the positions of size markers (in kDa).

View larger version (87K): [in a new window]   Fig. 2. Localisation of Ct-CRM1 and Ct-Ran in C. tentans cells. (A-C) Localisation of Ct-CRM1. Diploid tissue culture cells (A), and salivary gland cell (B). NE, nuclear envelope. The cells were immunostained with the anti-CRM1 antibody and a FITC-conjugated secondary antibody. (C) Immunoelectron microscope image of part of the nuclear membrane of a salivary gland cell. Gold particles (arrows) show the location of Ct-CRM1. Bar, 400 nm. (D-E) Localisation of Ct-Ran. (D) Tissue culture cells, showing mainly staining of the nuclei. (E) Salivary gland cells. Bars in A and C, 10 µm; bars in B and E, 20 µm.

View larger version (66K): [in a new window]   Fig. 7. Ct-CRM1 and Ct-Ran are associated with the BR mRNP particles during transport through the interchromatin. (A,B) BR mRNP particles (arrows) in the interchromatin labelled by Ct-CRM1-specific gold particles. (C,D) Ct-CRM1-labelled BR mRNP particles (arrows) docked at the NPCs. (E, F) BR mRNP particles (arrows) in the interchromatin, labelled by the anti-Ran antibody. (G,H) BR mRNP particles (arrows) docked at the NPCs, labelled by the anti-Ran antibody. To assist interpretation, schematic representations are shown below each electron micrograph. The labelled BR mRNP is shown in grey and the gold label in black. The nuclear membrane and the NPCs with their basket structure are outlined. Cyt, cytoplasm; Nu, nucleus. Bars, 100 nm.

View larger version (40K): [in a new window]   Fig. 3. Ct-CRM1 is associated with pre-mRNA at active gene loci in polytene chromosomes. (A) The three active BR gene loci on chromosome IV (arrows), and a few additional gene loci on the chromosome, were stained with the anti-CRM1 antibody. (B) Many gene loci on chromosome I were also stained by the antibody. (C, D) No staining of the gene loci was seen when a control antibody (anti-von Willebrand factor antibody) was used. (C',D') The chromosomes (in C and D) in phase contrast. (E,F) RNase treatment of the chromosomes before antibody staining abolished the staining of the gene loci. (E',F') Phase contrast images of the same chromosomes stained with the antibody in E and F; Bar, 10 µm. (G) Isolated chromosomes (chromosomes I and IV). Bar, 10 µm. (H) Western blot analysis of protein extract from tissue culture cells (lane 1) and the isolated chromosomes (seen in G) (lane 2). Both lanes were probed with anti-CRM1 and anti-Ct-eIF4H antibodies. Ct-eIF4H is known to be present mainly in the cytoplasm, but also in the interchromatin of the nucleus (Björk et al., 2003).

View larger version (102K): [in a new window]   Fig. 5. Ct-Ran is bound to chromatin, but also to nascent BR pre-mRNP. Anti-Ran staining of isolated chromosome IV (A) and chromosome I (B); Bar, 10 µm. In chromosome IV, the decondensed BR gene loci are only weakly stained (arrows). (C) Chromosome IV and III staining with the anti-Ran antibody is similar to staining of the chromosomal DNA with DAPI. In addition, the nucleolus on chromosome III was labelled. The two chromosomes are also shown in phase contrast. (D) RNase treatment before staining with the anti-Ran antibody did not significantly influence the staining pattern (except in the nucleolus). Bar, 10 µm. (E) Immunoelectron microscope analysis of Ct-Ran in a section through a BR gene. Gold labelling is seen of compact chromatin (Ch, thin arrows), of interchromatin regions between segments of transcribing BR genes (thick arrows), and of nascent BR pre-mRNP particles (arrowheads). Bar, 300 nm.

View larger version (42K): [in a new window]   Fig. 6. Immunoprecipitation of hnRNP complexes. Western blot of C. tentans nuclear proteins immunoprecipitated with the anti-hrp23 antibody. The blot was probed with a mixture of antibodies directed against hrp36 (similar to the mammalian hnRNP A/B proteins) (Visa et al., 1996), hrp23, a homologue of the Drosophila RSF1 protein (Labourier et al., 1999), CRM1 and Ran. Ct-CRM1, Ct-Ran, hrp36 and hrp23 were specifically detected (lane 3). A nuclear extract incubated with Protein A-Sepharose beads coupled to anti-mouse antibodies and anti-von Willebrand antibodies did not result in any precipitation of the proteins (lane 2). 1/80 of the nuclear extract used for immunoprecipitation is shown in lane 1. The bands migrating at approximately 55 kDa and 24 kDa were present in the negative control. In lane 3, the 24 kDa band is partly hidden by the Ct-Ran band.

View larger version (71K): [in a new window]   Fig. 8. Ct-CRM1 and Ct-Ran are translocated through the NPCs with the BR mRNP particles. Immunoelectron microscope labelling of sections through the nuclear membrane of salivary gland cells. (A-D) Anti-CRM1 labelling of BR mRNP particles (arrows) during progressive translocation through NPCs. (E-F) Anti-Ran labelling of BR mRNP particles (arrows) during translocation through NPCs. In the drawing below each micrograph, the BR mRNP is depicted in grey and the gold labelling in black. Cyt, cytoplasm; Nu, nucleus. Bars, 400 nm.

View larger version (82K): [in a new window]   Fig. 9. LMB treatment of C. tentans tissue culture cells does not influence the distribution of poly(A)+ RNA. Tissue culture cells, treated (B) or not treated (A) with LMB, were hybridised with an oligodT probe. No difference in the nuclear-cytoplasmic distribution of poly(A)+ RNA was seen. As a control for the effect of LMB, cells were stained for Dbp5 in the absence of LMB (C) and in the presence of LMB (D). Bar, 10 µm.

View larger version (61K): [in a new window]   Fig. 10. Effect of LMB treatment on salivary gland cells. C. tentans larvae were treated with LMB in water for 16 hours. Control animals were kept in water in parallel. Salivary glands were isolated, fixed and stained with anti-Dbp5 antibodies. In control glands (A, –LMB), Dbp5 is mainly seen in the cytoplasm and at the nuclear envelope. In LMB-treated glands (B, +LMB), Dbp5 is to a large extent present in the cell nuclei. Bar, 20 µm.

View larger version (115K): [in a new window]   Fig. 11. Treatment with LMB leads to an increase in the number of BR mRNP particles that are in transit through the NPCs. C. tentans larvae were treated with LMB and the salivary gland cells were analysed by electron microscopy. (A-D) Control cells without LMB. (A) BR mRNP particle docking at the NPC. (B-D) BR mRNP particles translocating through the NPC. (E-H) LMB-treated cells. (E) BR mRNP particle docking at the NPC. (F-H) BR mRNP particles translocating through the NPCs. Cyt, cytoplasm; Nu, nucleus. Bar, 100 nm.

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