Gle1 Regulates RNA Binding of the DEAD-Box Helicase Ded1 in Its Complex Role in Translation Initiation

Abstract

DEAD-box proteins (DBPs) are required in gene expression to facilitate changes to ribonucleoprotein complexes, but the cellular mechanisms and regulation of DBPs are not fully defined. Gle1 is a multifunctional regulator of DBPs with roles in mRNA export and translation. In translation, Gle1 modulates Ded1, a DBP required for initiation. However, DED1 overexpression causes defects, suggesting that Ded1 can promote or repress translation in different contexts. Here we show that GLE1 expression suppresses the repressive effects of DED1 in vivo and Gle1 counteracts Ded1 in translation assays in vitro Furthermore, both Ded1 and Gle1 affect the assembly of preinitiation complexes. Through mutation analysis and binding assays, we show that Gle1 inhibits Ded1 by reducing its affinity for RNA. Our results are consistent with a model wherein active Ded1 promotes translation but inactive or excess Ded1 leads to translation repression. Gle1 can inhibit either role of Ded1, positioning it as a gatekeeper to optimize Ded1 activity to the appropriate level for translation. This study suggests a paradigm for finely controlling the activity of DEAD-box proteins to optimize their function in RNA-based processes. It also positions the versatile regulator Gle1 as a potential node for the coordination of different steps of gene expression.

Keywords: DEAD box; RNA; helicase; nuclear export; translation; yeast.

FIG 1

GLE1 suppresses growth and translation inhibition caused by overexpression of DED1. (A) Wild-type (W303) cells transformed with Gal-inducible GLE1, DED1, and/or ded1-E307A were serially diluted, plated on selective medium containing galactose or dextrose, and incubated at 30°C. (B) Western blots (WB) of cell extracts from the strains described for panel A. Cells were grown in selective medium containing glucose and shifted to galactose-containing medium for 12 h before harvest. Samples were blotted using antibodies specific to Ded1, Gle1, and α-tubulin (Tub1). The relative intensity of the Ded1 bands is shown, normalized to Tub1, for three trials. (C to F) Cells transformed with Gal-inducible GLE1 and/or DED1 were shifted to galactose-containing medium for 12 h, and cell extracts were prepared. Polyribosomal profiles were generated by subjecting extracts to sucrose density centrifugation, followed by reading of the absorbance at 254 nM as the gradient was removed. Representative profiles are shown. Monosome-to-polysome (M/P) ratios were determined by calculating the area under the curve for the monosome (80S) peak versus the sum of the polysome peaks. Each M/P ratio presented is the mean from six independent trials ± standard error of the mean (SEM). **, P < 0.01 versus wild-type cells; ++, P < 0.01 versus DED1-overexpressing cells.

FIG 2

A balance of Ded1 and Gle1 activity is required for efficient translation and PIC assembly. (A and B) In vitro-transcribed luciferase mRNA was incubated with translation-competent extracts, followed by luciferase assays to determine the extent of translation. (A) Recombinant Ded1 was added to reaction mixtures with extracts from wild-type cells at 0.25, 0.5, 1, 2, 4, or 8 μM. (B) Ded1 (500 nM) and/or 250 nM recombinant Gle1 was added to translation-competent extracts from wild-type cells alone or in combination. *, P < 0.05 versus untreated (−); **, P < 0.01 versus untreated (−); +, P < 0.05 versus Ded1 sample. (C and D) Extracts from cells expressing protein A-tagged Ded1 were depleted of Ded1 with IgG-Sepharose resin or mock treated with glutathione-Sepharose. In vitro translation and luciferase assays were then performed with Gle1 added at 200 or 400 nM. (C) The activities in mock and depleted samples are directly compared. **, P < 0.01 versus mock; ++, P < 0.01 versus Ded1 depleted. (D) Activities in mock and depleted samples without Gle1 added are independently set to 100% in order to compare the responses to Gle1. *, P < 0.05 versus mock + 200 nM Gle1; +, P < 0.05 versus mock + 400 nM Gle1. For panels A to D, data represent the means from 3 to 7 independent experiments performed in duplicate. Error bars represent SEM. (E) In vitro-transcribed, ³²P-labeled MFA2p(G) mRNA was incubated with translation-competent extracts from wild-type cells in the presence of GMP-PNP to block initiation after 48S PIC assembly. Recombinant Gle1 (1 μM) and/or Ded1 (500 nM) was added as indicated. Samples were subjected to sucrose density fractionation, and radioactivity was quantitated by scintillation counting. The arrow marks the peak corresponding to the 48S PIC. Each data point represents the mean percentage at that fraction from three independent experiments. *, P < 0.05 versus untreated (−); +, P < 0.05 versus Ded1 sample.

FIG 3

Targeted mutations in ded1 affect suppression by GLE1. (A) In vitro pulldown assays were conducted with recombinant His-tagged Ded1 and a ded1-ΔNΔC deletion mutant (containing amino acids 126 to 538) from bacterial lysate. MBP-tagged Gle1 was incubated with samples as indicated. Samples were run on SDS-PAGE and Coomassie blue stained. (B) Effects on growth of the indicated mutants with or without GLE1 overexpression. Cells containing Gal-inducible DED1 (WT), ded1-N423A/F424A/R425A, ded1-N423A/F424A, ded1-Δ125-134, GLE1, and/or control plasmids were serially diluted on selective medium containing galactose or glucose and incubated at 30°C as described for Fig. 1A.

FIG 4

The GLE1-resistant ded1 mutants have defects in vivo and in vitro but still bind to Gle1. (A) Low-copy-number plasmids containing wild-type (WT) DED1, ded1-N423A/F424A/R425A, or ded1-N423A/F424A were introduced into ded1-null cells, and cells were serially diluted on yeast extract-peptone-dextrose (YPD) and grown at a range of temperatures. (B) Cells containing wild-type DED1 or ded1-N423A/F424A/R425A as the only copy of DED1 were grown at 30°C before temperature was shifted to 37°C for 60 min prior to harvest. Polysome profiles were then generated as described for Fig. 1B. *, P < 0.05 versus WT. (C) Cell extracts from the same conditions as described for panel B were blotted with antibodies to Ded1 and PGK1 (loading control). Note that wild-type Ded1 levels decrease slightly at 37°C. (D) In vitro pulldown assays were performed with His-tagged, recombinant wild-type Ded1, ded1-N423A/F424A, and ded1-Δ125-134. Untagged Gle1 was incubated with samples as indicated. The proteins alone are shown in the “inputs” lanes (right). Samples were run on SDS-PAGE and Coomassie blue stained. (E) ATPase activity of purified recombinant wild-type Ded1, ded1-N423A/F424A/R425A, ded1-N423A/F424A, and ded1-Δ125-134 was determined with a colorimetric PK/LDH-coupled assay in the absence or presence of total cellular RNA (10 μg/ml). Values shown are the means from 4 independent experiments performed in duplicate.

FIG 5

Gle1 inhibits the RNA binding activity of Ded1. (A to D) Equilibrium RNA binding assays were performed with wild-type Ded1, ded1-N423A/F424A, and ded1-Δ125-134. Filter binding onto nitrocellulose and nylon was assessed with radiolabeled RNA oligonucleotides incubated with recombinant protein in the presence of no additional nucleotide (apo) (A), ADP-BeF_x (B), or ADP (C). (D) Apparent binding affinities under the different conditions were calculated via a single-binding-site model. (E) Fluorescence anisotropy was used to assess the RNA binding affinity of Ded1 in the presence of ADP-BeF_x with or without 100 nM Gle1. Curve-fitting and apparent binding affinities were determined as described above.