mRNA Export from Mammalian Cell Nuclei Is Dependent on GANP

Summary Bulk nuclear export of messenger ribonucleoproteins (mRNPs) through nuclear pore complexes (NPCs) is mediated by NXF1. It binds mRNPs through adaptor proteins such as ALY [1, 2] and SR splicing factors [3] and mediates translocation through the central NPC transport channel via transient interactions with FG nucleoporins [4–10]. Here, we show that mammalian cells require GANP (germinal center-associated nuclear protein) for efficient mRNP nuclear export and for efficient recruitment of NXF1 to NPCs. Separate regions of GANP show local homology to FG nucleoporins, the yeast mRNA export factor Sac3p, and the mammalian MCM3 acetyltransferase. GANP interacts with both NXF1 and NPCs and partitions between NPCs and the nuclear interior. GANP depletion inhibits mRNA export, with retention of mRNPs and NXF1 in punctate foci within the nucleus. The GANP N-terminal region that contains FG motifs interacts with the NXF1 FG-binding domain. Overexpression of this GANP fragment leads to nuclear accumulation of both poly(A)+RNA and NXF1. Treatment with transcription inhibitors redistributes GANP from NPCs into foci throughout the nucleus. These results establish GANP as an integral component of the mammalian mRNA export machinery and suggest a model whereby GANP facilitates the transfer of NXF1-containing mRNPs to NPCs.

(same buffer + 1 % BSA), coverslips were incubated with the appropriate primary and secondary antibodies (Molecular Probes) and examined using a Zeiss LSM510 Meta confocal microscope. For some experiments, cells were permeabilised prior to fixation with PBS, 0.1 % Triton X-100 for 5 mins on ice. For some experiments, cells were treated with Actinomycin D (5 μg/ml) or DRB (50 μg/ml) (Sigma) for between 2 to 4 hours. For antibody access experiments, cells were permeabilised after fixation with 0.004 % digitonin (ICN Biomedicals) for 5 mins on ice, or with 0.2 % Triton X-100 for 5 mins at room temperature. Scanning analysis of cells was performed using ImageJ software (NIH). All images used for comparative analysis were acquired using identical microscope settings. A line width of 20 was used, and pairs of cells with nuclei of same scan width as indicated by DAPI staining were used for analysis. All analyses are representative of the cell population.
Transfections with plasmid DNA where indicated were carried out with Genejuice as per manufacturers instructions (Novagen).
Briefly, 10 x 10 6 cells were permeabilised with 10 mls of 50 μg/ml digitonin (ICN Biomedicals), 100 mM NaCl, 10 mM Tris pH 8.0 and protease inhibitors (Roche) for 10 mins on ice. Following centrifugation at 800 g for 3 mins, pelleted nuclei were washed with 10 mM Tris pH 8.0, 100 mM NaCl, protease inhibitors and nuclei were lysed with 400 μl of 10 mM Tris pH 8.0, 500 mM NaCl, 0.1 % NP-40 and protease inhibitors for 10 mins on ice. Following centrifugation at 14000 rpm for 30 mins at 4 0 C, supernatant was diluted in lysis buffer without NaCl so that final concentration of NaCl was 150 mM. For some samples, RNase (0.2 mg/ml) was added to nuclei lysis buffer and all subsequent buffers. Samples were then processed as above.
Cultures were grown at 37 0 C to an OD 600 of 0.6, then induced by addition of IPTG to 1mM and grown at 30 or 37 0 C for 4 hours. Cells expressing GST-GANP(1-313) were lysed in 1xPBS, protease inhibitors (Roche), and sonicated on ice. 1 % Triton X-100 (Sigma) was added and incubated for 30 mins at 4 0 C. GANP fragment was bound to Glutathione Sepharose (GE Healthcare) for 2 hours at 4 0 C with rotation, and beads washed extensively to remove unbound proteins. GST-GANP(1-313) was eluted with 50 mM Tris-HCl pH 8.0, 20 mM reduced glutathione. Cells expressing His-NXF1 (371-621) were lysed in lysis buffer (50 mM phosphate buffer, 300 mM NaCl, 10 mM imidazole, pH 8.0), and lysozyme added for 30 mins at 4 0 C. After sonication on ice, lysate was centrifuged at 10 000 g for 25 mins at 4 0 C. Cleared lysate was purified over Ni-Nta agarose (Qiagen) and washed extensively with lysis buffer with 20 mM imidazole, followed by lysis buffer with 40 mM imidazole. Binding assays were performed by immobilising His-NXF1(371-621) on Ni-Nta agarose resin. Purified GST or GST-GANP(1-313) was incubated with resin for 2hrs at 4 0 C and washed extensively with 50 mM phosphate buffer pH 8.0, 150 mM NaCl, 10 mM imidazole. Samples were analysed by SDS-PAGE.

RNA FISH
RNA FISH was performed as previously described [1] using an oligo(dT) primer (Sigma). Nuclear accumulation of poly(A)+ RNA in GANP depleted cells was quantitated and compared to cells transfected with the control siRNA. At least 1400 nuclei per sample were analysed using ScanR acquisition and analysis software (Olympus). Nuclear Cy3 intensity and standard deviation were calculated for each coverslip. Nuclear Cy3 intensity was measured for the whole nucleus and not adjusted for the unstained nucleoli.
Also, nuclei were obtained by digitonin permeabilisation as above, and nuclei were lysed as above to obtain soluble nuclear extract for IP. Samples were analysed by SDS-PAGE followed by immunoblotting with anti-NXF1 (Abcam) as positive control, anti-GANP, and anti-MCM2 (BD Biosciences) as negative control.

1.
Herold   Table  representing identity (%) and similarity (%) between protein sequences of a region of GANP (Human, 634-936, and mouse), Sac3p (S.cerevisiae) and Xmas-2 (Drosophila) based on GANP protein alignment with Sac3p and Xmas-2 using ClustalW and Boxshade. Black boxes represent identity, grey boxes represent similarity. (D) Characterisation and validation of GANP antibody. Anti-GANP immunofluorescence was carried out on U20S cells with or without antigen and also on GANP depleted cells. Secondary antibody only control is shown. Nuclei were stained with DAPI. (E) GANP is localised to the nuclear face of the NPC. HCT116 cells were permeabilised with either digitonin, which preferentially permeabilises the plasma membrane, or Triton X-100, which permeabilises both plasma and nuclear membranes, and stained with anti-GANP, Lamin B and Nup358 antibodies. The integrity of the nuclear and plasma membranes following digitonin or Triton X-100 permeabilisation is confirmed by staining of Nup358, a nucleoporin that is localised to the cytoplasmic face of the NPC, of Lamin B, an inner nuclear membrane protein and of β−actin, a cytoplasmic protein. Sac3p  Xmas-2 (201-560) (2 out of 3) (2 out of 3) Sac3p  Sac3p     Figure 2A. Coverslips were analysed for nuclear Cy3 intensity using ScanR acquisition and analysis software (Olympus). Nuclear Cy3 intensity was measured for the whole nucleus and not adjusted for the unstained nucleoli. A plot of counts vs. nuclear Cy3 intensity is shown. Black represents GANP siRNA treated cells, and red represents control siRNA treated cells. Nuclear Cy3 intensity and standard deviation are shown for each population in the table. For ScanR analysis, cells were synchronised in S-phase prior to harvest. At least 1400 nuclei were counted for each coverslip. (B) NPC is functional in GANP depleted cells. Nuclear import and CRM-1 dependent export of endogenous STAT2 proceed in the absence of GANP. Anti-STAT2 immunofluorescence was carried out on HeLa cells transfected with control siRNA or GANP siRNA and treated with leptomycin B (20 nM) for 1 hour to inhibit CRM-1 dependent export.  Nuclear Interior Figure S4-GANP localisation is altered following treatment with Actinomycin D HCT116 cells were treated with transcription inhibitor Actinomycin D for 4 hours and immunofluorescence was performed using anti-GANP and anti-NXF1 antibodies respectively. Scanning analysis of GANP intensity in untreated or Actinomycin D treated HCT116 cells using ImageJ software was performed. Nuclear envelope (NE) and nuclear interior are indicated.