Control of mRNA export and translation termination by inositol hexakisphosphate requires specific interaction with Gle1

Abstract

The unidirectional translocation of messenger RNA (mRNA) through the aqueous channel of the nuclear pore complex (NPC) is mediated by interactions between soluble mRNA export factors and distinct binding sites on the NPC. At the cytoplasmic side of the NPC, the conserved mRNA export factors Gle1 and inositol hexakisphosphate (IP(6)) play an essential role in mRNA export by activating the ATPase activity of the DEAD-box protein Dbp5, promoting localized messenger ribonucleoprotein complex remodeling, and ensuring the directionality of the export process. In addition, Dbp5, Gle1, and IP(6) are also required for proper translation termination. However, the specificity of the IP(6)-Gle1 interaction in vivo is unknown. Here, we characterize the biochemical interaction between Gle1 and IP(6) and the relationship to Dbp5 binding and stimulation. We identify Gle1 residues required for IP(6) binding and show that these residues are needed for IP(6)-dependent Dbp5 stimulation in vitro. Furthermore, we demonstrate that Gle1 is the primary target of IP(6) for both mRNA export and translation termination in vivo. In Saccharomyces cerevisiae cells, the IP(6)-binding mutants recapitulate all of the mRNA export and translation termination defects found in mutants depleted of IP(6). We conclude that Gle1 specifically binds IP(6) and that this interaction is required for the full potentiation of Dbp5 ATPase activity during both mRNA export and translation termination.

FIGURE 1.

Dbp5 stimulates binding of Gle1 and IP₆. A, equilibrium binding assays utilizing 10 nm [³H]IP₆ and Gle1 (solid line) or Gle1 + 1 μm Dbp5 (dashed line) were used to determine the dissociation constant (K_d) of Gle1 for IP₆. 19.5 cpm = 1 fmol of IP₆ bound. B, equilibrium binding assays as in A were used to measure the stimulation of Dbp5 for Gle1-IP₆ binding using 25 nm Gle1 and 10 nm [³H]IP₆. Gle1-IP₆ binding in the absence of Dbp5 was subtracted out to achieve the saturation curve shown. C, equilibrium binding assays as in A were used to test the effect of known regulators of Dbp5 activity on Gle1-IP₆ binding. 10 nm [³H]IP₆, 25 nm Gle1, and 100 nm Dbp5 were used for these assays. **, p < 0.01 versus Gle1 + Dbp5; ++, p < 0.01 versus Gle1 + Dbp5 + AMP-PNP by Student's t test. The mean and standard error of the mean were calculated from three to eight independent experiments in A–C.

FIGURE 2.

Gle1 residues Lys³⁷⁷ and Lys³⁷⁸ are required for the IP₆-mediated Dbp5 ATPase stimulation. A, sequence alignment of conserved regions in the C-terminal domain of Gle1 from selected fungal and metazoan species. Residues with greater homology identified by a modified Clustal-W alignment (see “Experimental Procedures”) are depicted in increasingly darker gray. The asterisks denote amino acids selected as putative IP₆ binding residues. B, bacterially expressed, purified recombinant Gle1, gle1-K377Q, gle1-K377Q/K378Q, and gle1-K494Q were separated in a 7.5% SDS-polyacrylamide gel and stained with Coomassie Blue. C, equilibrium binding assays utilizing 10 nm [³H]IP₆ and increasing amounts of Gle1 were used to calculate the K_d of Gle1 proteins for IP₆. D, equilibrium binding assays as shown in C in the presence of 1 μm Dbp5. E, Dbp5 ATPase assays were conducted utilizing 100, 400, or 800 nm of Gle1, 1 mm ATP, 10 μm RNA, and 200 nm Dbp5. 100 nm IP₆ was added to each sample containing 800 nm Gle1. The mean and standard error of the mean were calculated from three independent experiments in C–E.

FIGURE 3.

Protein stability, localization, and growth phenotype of strains expressing gle1 mutant alleles. A, indicated strains were grown at 23 °C and shifted to 30 and 37 °C for 1 h prior to immunoblot analysis utilizing anti-Gle1 polyclonal antibodies. Anti-Pgk1 was used as a loading control. B, Gle1 localization was observed by indirect immunofluorescence microscopy utilizing anti-Gle1 polyclonal antibodies, and the nuclei were visualized with DAPI. gle1Δ mutant strains carrying plasmids harboring GLE1, gle1-K377Q, gle1-K377Q/K378Q, or gle1-K494Q were grown at 23 °C and shifted to 30 and 37 °C for 1 h prior to processing. C, indicated strains were spotted in 5-fold serial dilutions and incubated in rich medium at 23, 30, and 37 °C.

FIGURE 4.

In vitro IP₆ binding defects correlate with mRNA export and translation termination defects in vivo. A, an ipk1Δ strain and gle1Δ mutant strains carrying plasmids harboring either GLE1, gle1-K377Q, gle1-K377Q/K378Q, or gle1-K494Q were used for in situ hybridization with a digoxigenin-coupled oligo(dT) probe after growing at 23 °C and shifting to 30 or 37 °C for 1 h. Poly(A)⁺ RNA localization was visualized by indirect immunofluorescence microscopy with a fluorescein isothiocyanate-coupled anti-digoxigenin antibody, and the nuclei were visualized with DAPI staining as indicated. B, quantification of cells in A presenting nuclear mRNA accumulation. The bars represent the percentages of cells with mRNA export defects from a total of 50–130 cells/condition. C, wild type (W303), gle1-4, ipk1Δ, rat8-2 (dbp5), gle1-K377Q, and gle1-K377Q/K378Q strains transformed with TQ/U and TMV/U tandem reporter constructs were grown overnight at 23 °C and shifted to 37 °C for 30 min prior to harvest. Luciferase and β-galactosidase assays were performed, and the ratios of the activities were calculated. Read-through activity is expressed as the percentage of the TMV reporter compared with TQ control for each strain. The mean and standard error of the mean were calculated from three to eight independent experiments. *, p < 0.05; **, p < 0.01 by Student's t test.