Depletion of a single nucleoporin, Nup107, prevents the assembly of a subset of nucleoporins into the nuclear pore complex

Abstract

The nuclear pore complex (NPC) is a protein assembly that contains several distinct subcomplexes. The mammalian nucleoporin (Nup)-107 is part of a hetero-oligomeric complex, that also contains Nup160, Nup133, Nup96, and the mammalian homolog of yeast Sec13p. We used transfection of HeLa cells with small interfering RNAs to specifically deplete mRNA for Nup107. In a domino effect, Nup107 depletion caused codepletion of a subset of other Nups on their protein but not on their mRNA level. Among the affected Nups was a member of the Nup107 subcomplex, Nup133, whereas two other tested members of this complex, Nup96 and Sec13, were unaffected and assembled into Nup107Nup133-deficient NPCs. We also tested several phenylalanine-glycine repeat-containing Nups that serve as docking sites for karyopherins. Some of these, such as Nup358, Nup214 on the cytoplasmic, and Nup153 on the nucleoplasmic side of the NPC, failed to assemble into Nup107Nup133-depleted NPCs, whereas p62, a Nup at the center of the NPC, was unaffected. Interestingly, the filamentous, NPC-associated protein Tpr also failed to assemble into the NPCs of Nup107-depleted cells. These data indicate that Nup107 functions as a keystone Nup that is required for the assembly of a subset of Nups into the NPC. Despite the depletion of Nup107 and the accompanying effects on other Nups, there was no significant effect on the growth rate of these cells and only a partial inhibition of mRNA export. These data indicate redundancy of Nups in the function of the mammalian NPC.

Figure 1

Effects of Nup107-specific RNAi on mRNA and protein levels of Nup107 and Nup96. (A) mRNA levels of Nup107 and Nup96 were determined by RT-PCR in nontransfected HeLa cells (−) or in HeLa cells 24 or 48 h after a single transfection (+) with Nup107-specific siRNAs. For the 72-h time point, a second transfection was applied 36 h after the initial transfection (++). The Nup107 mRNA level in the cells transfected with Nup107 siRNAs was significantly reduced at each time point, whereas the mRNA level of Nup96 was not affected at any time point. (B) Lysates of HeLa cells, either nontransfected (Left) or 48 h after transfection with mock (Middle) or Nup107-specific siRNAs (Right), were immunoblotted with α-Nup107 (Upper) and α-Nup96 antibodies (Lower). Cells transfected with Nup107 siRNAs showed a significant reduction of Nup107, whereas Nup96 was not affected.

Figure 2

Immunofluorescence analysis of Nup107 depletion. HeLa S3 cells, double-labeled with α-Nup107 antibodies and mAb414, were analyzed by immunofluorescence microscopy. Nontransfected cells displayed nuclear rim staining with both antibodies (A–C). Nup107 was dramatically reduced at the nuclear rim of cells 48 h after transfection with Nup107-specific siRNAs (D and F). The five cells in the upper part of D and F displaying nuclear rim staining with α-Nup107 represented nontransfected cells. Note that the Nup107-depleted cells also exhibited reduced labeling intensity with mAb414 (E and F).

Figure 3

Effects of Nup107 depletion on protein and mRNA levels of other NPC-associated proteins. (A) Lysates of nontransfected HeLa cells (−) and of HeLa cells 48 h after transfection with Nup107 siRNAs (+) were analyzed by immunoblotting with the indicated antibodies. Nup133, Nup358, Nup214, Nup153, and Tpr were significantly reduced in the Nup107-depleted samples, whereas Nup96, Sec13, and p62 were not affected. (B) The corresponding total RNA extracts were analyzed by RT-PCR. Only mRNA for Nup107 was depleted.

Figure 4

Immunofluorescence analysis of other members of the Nup107 subcomplex after Nup107 depletion. HeLa S3 cells, double-labeled with indicated antibodies, were analyzed 48 h after transfection. Nontransfected cells displayed nuclear rim staining with either α-Nup133 antibodies or mAb414 (A–C). Whereas the signal of Nup133 was greatly reduced at the nuclear rim of Nup107-depleted cells (D and F), there was no change in Nup96 labeling in Nup107-depleted cells (G and J) when compared with the two nontransfected cells in H. Nup107-depleted cells showed reduced labeling with mAb414 (E and H).

Figure 5

Immunofluorescence analysis of FG-containing Nups after Nup107 depletion. HeLa S3 cells, double-labeled with indicated antibodies, were analyzed 48 h after transfection. Compared with nontransfected cells (A–C), Nup358 labeling was reduced at the nuclear rim and increased in the cytoplasm in Nup107-depleted cells (D and F), whereas labeling with α-p62 exhibited no significant change (G and J). Nup107-depleted cells showed reduced labeling with mAb414 (E and H).

Figure 6

Nuclear accumulation of poly(A)⁺ RNA in Nup107-depleted cells. Localization of poly(A)⁺ RNA was determined by in situ hybridization with biotinylated oligo-dT and indirect fluorescence microscopy. Nontransfected HeLa cells (G–J) and cells 72 h after double transfections with Nup107-specific (A–C) or mock siRNAs (D–F) were analyzed. The cells were immunolabeled with α-Nup107 antibodies. In comparison to mock-transfected (D–F) or nontransfected cells (G–J), the Nup107-depleted cells exhibited nuclear accumulation of poly(A)⁺ RNA (A–C).