The human homologue of Saccharomyces cerevisiae Gle1p is required for poly(A)+ RNA export

Abstract

The mechanism of mRNA export is a complex issue central to cellular physiology. We characterized previously yeast Gle1p, a protein with a leucine-rich (LR) nuclear export sequence (NES) that is essential for poly(A)+ RNA export in Saccharomyces cerevisiae. To characterize elements of the vertebrate mRNA export pathway, we identified a human homologue of yeast Gle1p and analyzed its function in mammalian cells. hGLE1 encodes a predicted 75-kDa polypeptide with high sequence homology to yeast Gle1p, but hGle1p does not contain a sequence motif matching any of the previously characterized NESs. hGLE1 can complement a yeast gle1 temperature-sensitive export mutant only if a LR-NES is inserted into it. To determine whether hGle1p played a role in nuclear export, anti-hGle1p antibodies were microinjected into HeLa cells. In situ hybridization of injected cells showed that poly(A)+ RNA export was inhibited. In contrast, there was no effect on the nuclear import of a glucocorticoid receptor reporter. We conclude that hGle1p functions in poly(A)+ RNA export, and that human cells facilitate such export with a factor similar to yeast but without a recognizable LR-NES. With hGle1p localized at the nuclear pore complexes, hGle1p is positioned to act at a terminal step in the export of mature RNA messages to the cytoplasm.

Figure 1

Amino acid sequence of hGle1p. (A) hGle1p amino acid sequence. (B) Coiled-coil probability for hGle1p. A middle region of hGle1p shows a high probability of forming a coiled-coil structure by analysis with the coils program (48). (C) Comparison of the C-terminal regions of yeast and human Gle1p. align analysis (43) between the yeast (upper line) and human proteins (lower line) reveals significant homology. The center line designates identical (capital letter) and conserved (: or .) residues. The LR-NES in yeast Gle1p is boxed. The slash (/) designates the breakpoint for Regions 3 and 4 (Fig. 3A). The plus signs (+) mark residues changed in yeast gle1 alleles (P380G or G384R).

Figure 2

hGle1p localizes to the NPC and is cytoplasmically accessible. (A) Immunoblot analysis with whole-cell HeLa extracts and affinity-purified rabbit antibodies: anti-hGle1p (lane 1) or anti-MBP (lane 2). Molecular mass markers are in kDa. (B–K) Intracellular distribution of hGle1p. HeLa cells were processed for labeling with primary antibodies as designated: Fix-Triton (B and C), Digitonin-Fix-Triton (D, E, and H–J), or Digitonin-Fix (F, G, and K). hGle1p localization was observed with the affinity-purified rabbit anti-hGle1p antibody (B–G), nucleoporin staining (H and I) with mAb414 (40), and lamin staining with a rabbit anti-lamin B serum (J and K) (41). Pairs of cells (B–C, D–E, H–I, and F–G) were photographed in two different focal planes for the punctate (C, D, H, and F) and nuclear rim (B, E, G, and I) staining. (Bars = 10 μm.)

Figure 3

Complementation analysis of yeast gle1–4 cells with hGLE1 chimeras. (A) Schematic representation of the structural regions in yeast (red) and human (blue) Gle1p proteins, with the LR-NES designated (yellow). (B) Yeast gle1–4 strains expressing the designated yeast deletion (Δ) or yeast–human chimeric Gle1p were grown at 23°C (Right) or 30°C (Left). Serial dilutions (from left to right) of equivalent cell numbers were plated on synthetic minimal medium lacking leucine. Expression of full-length yeast Gle1p supports growth at 30°C (top row), whereas vector alone does not (second row). Immunoblot analysis confirmed expression of all polypeptides (data not shown).

Figure 4

Injection of anti-hGle1p antibodies inhibits poly(A)⁺ RNA export in HeLa cells. The distribution of poly(A)⁺ RNA was examined in HeLa cells co-microinjected with Texas red–dextran and either affinity-purified rabbit anti-hGle1p antibody alone (2.7 mg/ml, Left) or anti-hGle1p premixed with purified GST-hGle1p (2.7 mg/ml IgG and 2.25 mg/ml recombinant, Right). The anti-hGle1p antibody is monospecific for hGle1p (see Fig. 2A). Only a subset of the cells in a given field were injected, and all the cells were cytoplasmically injected except for the uppermost injected cell in the Right column (by Texas red localization, Middle). Cells were incubated for 12 hr at 37°C before in situ hybridization with a digoxigenin-labeled oligo(dT₃₀) probe and fluorescein isothiocyanate antidigoxigenin Fab. Arrowhead highlights a typical injected cell, and arrow indicates an uninjected cell (Top). Nuclear DNA was stained with 4′,6-diamidino-2-phenylindole (Bottom). (Bar = 20 μm.)

Figure 5

Nuclear import is not affected by microinjection of anti-hGle1p antibody. HeLa cells transiently expressing GR-GFP (A, D, G, and J) were co-microinjected with Texas red–dextran and either affinity-purified rabbit antibody: anti-hGle1p (2 mg/ml, A–F) or anti-MBP (2 mg/ml, G–L). The anti-MBP antibody served as a control for nonspecific effects of antibody injection. Texas red–dextran (Center, B, E, H, and K) differentiates between injected and uninjected cells. In a given field, only a subset of the cells were cytoplasmically injected. Cells were incubated for 12 hr at 37°C, and those in D–F and J–L were treated with medium containing 10 μg/ml dexamethasone for 30 min before fixation and processing for fluorescence microscopy. Cells in A–C and G–I were untreated. Nuclear DNA was stained with 4′,6-diamidino-2-phenylindole (C, F, I, and L). (Bar = 20 μm.)