The mRNA export factor Gle1 and inositol hexakisphosphate regulate distinct stages of translation

Abstract

Gene expression requires proper messenger RNA (mRNA) export and translation. However, the functional links between these consecutive steps have not been fully defined. Gle1 is an essential, conserved mRNA export factor whose export function is dependent on the small molecule inositol hexakisphosphate (IP(6)). Here, we show that both Gle1 and IP(6) are required for efficient translation termination in Saccharomyces cerevisiae and that Gle1 interacts with termination factors. In addition, Gle1 has a conserved physical association with the initiation factor eIF3, and gle1 mutants display genetic interactions with the eIF3 mutant nip1-1. Strikingly, gle1 mutants have defects in initiation, whereas strains lacking IP(6) do not. We propose that Gle1 functions together with IP(6) and the DEAD-box protein Dbp5 to regulate termination. However, Gle1 also independently mediates initiation. Thus, Gle1 is uniquely positioned to coordinate the mRNA export and translation mechanisms. These results directly impact models for perturbation of Gle1 function in pathophysiology.

Figure 1. A role for Gle1 in translation

(A) Cultures of gle1-2, gle1-4, rat7-1 (nup159), nup42Δ, gfd1Δ, and wild-type (WT) strains were spotted in five-fold serial dilutions on YPD alone or YPD containing 0.1 μg/ml cycloheximide or 0.4 mg/ml paromomycin, then incubated 2 days at 23°C. (B) Cultures of gle1-4 and wild-type strains transformed with empty URA3/CEN plasmid (pURA3) or a GLE1/URA3/CEN (pGLE1) plasmid were spotted as in (A) on Ura⁻ selective media with or without cycloheximide. (C) Lysates of wild-type strains grown at 23°C were subjected to sucrose density fractionation and immunoblotted with α-Gle1 and α-Rps6 (ribosomal protein control). Ribosome distribution was determined by OD₂₅₄.

Figure 2. Gle1 interacts with termination factors

(A) Cultures of gle1-4, sup45-2, gle1-4 sup45-2, and wild-type control (WT) strains were serially diluted, spotted on YPD, and incubated 2–3 days at 23°or 30°C. (B) Immunoprecipitations were performed with lysates from SUP35-TAP, SUP45-TAP, and wild-type (WT) yeast strains. Lysates (10% of input) and total immunoprecipitates (IP) were immunoblotted with α-mouse IgG (to detect TAP-tagged proteins (ProtA) or α-Gle1. (C) Soluble binding assays were performed with GST-Sup45 or GST from bacterial lysates and recombinant purified Gle1. Gle1 input (10%) and bound samples were immunoblotted with α-GST or α-Gle1.

Figure 3. Gle1 is required for efficient translation termination

(A) Cultures of gle1-2, sup45-2, gle1-2 sup45, gle1-4 sup45-2 and wild-type controls (WT) were serially diluted and spotted on 0.25 YPD. Plates were incubated 4 days at 23°C and colony color assessed. (B) The tandem β-galactosidase/luciferase reporter constructs are shown: TQ (control) lacks a stop codon in the linker, TMV has a stop codon inserted in frame into the linker region, and 1789 has a stem-loop from HIV-1 inserted into the linker. (C) gle1-2, gle1-4, rat8-2 (dbp5), rat7-1 (nup159), and wild-type control (WT) strains were transformed with the tandem reporter constructs and luciferase and β-galactosidase assays were performed. Activities were normalized to cell number, and ratios of luciferase to β-galactosidase activity determined. Read-through activity is expressed as the percentage of the TMV or 1789 reporter compared to TQ control for each strain, n = 3–5 independent experiments. Error bars = 1 SEM above and below the mean. ** p<0.01 (D) Lysates of SUP35-TAP and gle1-4 SUP35-TAP strains grown at 23°C were subjected to sucrose density fractionation and immunoblotted with α-mouse IgG to detect Sup35-TAP. Ribosome distribution was determined by OD₂₅₄, and blots were analyzed by densitometry to assign ribosome association percentages (n = 4). Error = 1 SEM above and below the mean. p<0.05

Figure 4. Gle1 has conserved interactions with eIF3 subunits

(A) Plasmids expressing hGle1-GFP and eIF3f-HA were cotransfected into HeLa cells, and immunoprecipitations were performed using α-GFP antibodies. Lysates (5%) and immunoprecipitates were immunoblotted using α-GFP or α-HA. (B) Immunoprecipitations were performed with lysates from PRT1-TAP, NIP1-TAP, and wild-type (WT) yeast strains. Lysates (10% of input) and total immunoprecipitates (IP) were immunoblotted with α-mouse IgG (for TAP-tagged proteins (ProtA)) or α-Gle1. (C) Recombinant purified Gle1 was incubated with bacterial lysates for GST-Prt1, -Nip1, -Rpg1/Tif32, -Tif34, or -Tif35, and bound proteins were isolated by immunoprecipitation with α-Gle1. In controls, lysates were incubated without Gle1. RNase A treatment was conducted with the Gle1/GST-Prt1 sample. Total bound and input (5%) were immunoblotted with α-GST or α-Gle1. (D) Cultures of the respective gle1 and nip1 single and double mutant strains with wild-type controls (WT) were serially diluted and spotted on YPD. Plates were incubated 3 days at 23°C, 30°C or 37°C.

Figure 5. Strains harboring gle1 mutant alleles have polysome profile defects

Polysome profiles were generated by subjecting cell lysates to 7–47% sucrose density centrifugation and OD₂₅₄ analysis of the fractionated gradient. The monosome (80S) and polysome peaks are labeled in (A). Cultures of wild-type cells were grown at 23°C (A) or shifted to 37°C for 75 min prior to harvest (B). Cultures of gle1-4 were grown at 23°C (C) or shifted to 37°C for 75 min prior to harvest (D). The mRNA export mutants gle2Δ (E) and nup116Δ (F) were shifted to 37°C for 75 min prior to harvest. Note that both monosome and polysome peaks are reduced in the nup116Δ strain. The gle1-4 and wild-type control (WT) strains harboring either a URA3/CEN or GLE1/URA3/CEN plasmid were shifted to 37°C for 60 min prior to harvest (G–J).

Figure 6. GCN4 activation defects are detected in gle1 mutants

Wild-type (WT), gle1-2, gle1-4, rat7-1 (nup159), and rat8-2 (dbp5) strains were transformed with a GCN4-lacZ reporter plasmid. Cultures were grown in Ura⁻ His⁻selective media at either 23°C or 30°C for 2h. 3-aminotriazole (3AT) was added as indicated. Following growth overnight at 23°C or 30°C, assays for β-galactosidase activity were performed. Activity is expressed as β-gal units (1 unit hydrolyzes 1 μmol of ONPG substrate per min per cell). Error bars = 1 SEM above and below the mean. Table summarizes fold activation (+3AT divided by −3AT) for each strain at each temp. * p<0.05 for the fold activation at 23°C vs. 30°C. (n = 3–5 independent experiments).

Figure 7. IP₆ regulates termination but not initiation

(A) Cultures of ipk1Δ and wild-type (WT) strains were spotted in five-fold serial dilutions on YPD alone or on YPD containing 0.1 μg/ml cycloheximide. Plates were incubated 2 days at 23°C. (B) Cultures of ipk1Δ, sup45-2, ipk1Δ sup45-2 and wild-type (WT) strains were serially diluted, spotted on YPD, and incubated 2 days at 23°C, 30°C, 34°C, or 37°C. (C) Read-through assays were performed with ipk1Δ, ipk2Δ, vip1Δ, kcs1Δ, and wild-type control (WT) strains using the β-galactosidase/luciferase tandem reporters. Read-through efficiency was determined as in Figure 3C, n = 4–6 independent experiments. (D) Polysome profiles for ipk1Δ were generated as in Figure 5. Cells were shifted to 37°C for 75 min prior to harvest. (E) GCN4 activation assays were performed with ipk1Δ and wild-type control (WT) strains using the GCN4-lacZ reporter as in Figure 6.