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. 2020 Dec 4;295(49):16813-16825.
doi: 10.1074/jbc.RA120.015715. Epub 2020 Sep 27.

Functions of Gle1 are governed by two distinct modes of self-association

Aaron C Mason  1 , Susan R Wente  2
Affiliations

Functions of Gle1 are governed by two distinct modes of self-association

Aaron C Mason et al. J Biol Chem. .

Abstract

Gle1 is a conserved, essential regulator of DEAD-box RNA helicases, with critical roles defined in mRNA export, translation initiation, translation termination, and stress granule formation. Mechanisms that specify which, where, and when DDXs are targeted by Gle1 are critical to understand. In addition to roles for stress-induced phosphorylation and inositol hexakisphosphate binding in specifying Gle1 function, Gle1 oligomerizes via its N-terminal domain in a phosphorylation-dependent manner. However, a thorough analysis of the role for Gle1 self-association is lacking. Here, we find that Gle1 self-association is driven by two distinct regions: a coiled-coil domain and a novel 10-amino acid aggregation-prone region, both of which are necessary for proper Gle1 oligomerization. By exogenous expression in HeLa cells, we tested the function of a series of mutations that impact the oligomerization domains of the Gle1A and Gle1B isoforms. Gle1 oligomerization is necessary for many, but not all aspects of Gle1A and Gle1B function, and the requirements for each interaction domain differ. Whereas the coiled-coil domain and aggregation-prone region additively contribute to competent mRNA export and stress granule formation, both self-association domains are independently required for regulation of translation under cellular stress. In contrast, Gle1 self-association is dispensable for phosphorylation and nonstressed translation initiation. Collectively, we reveal self-association functions as an additional mode of Gle1 regulation to ensure proper mRNA export and translation. This work also provides further insight into the mechanisms underlying human gle1 disease mutants found in prenatally lethal forms of arthrogryposis.

Keywords: DEAD-box protein; Gle1; mRNA; mRNA export; nuclear pore; oligomerization; protein aggregation; stress granules; translation regulation.

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Conflict of interest statement

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Coiled-coil domain contributes to Gle1 oligomerization.A, human Gle1 domain organization schematic. B, MARCOIL predicted coiled-coil probability of Gle1. C, assessment tool for homodimeric Coiled-Coil ORientation Decision (ACCORD): size-exclusion chromatography with multiangle light scattering (SEC-MALS) of SspB-Gle1152–360 and BSA. SEC-MALS experiments were performed in buffer containing 20 mm HEPES (pH 7.6), 200 mm NaCl, 5% glycerol, 0.5 mm TCEP, 0.05% azide with a Superose 6 10/300 GL column connected to a Wyatt Dawn8+ system. D, predicted putative heptad repeat of the Gle1 coiled-coil domain. Heptad repeats with a probability greater than 90% are shown. Bold letters indicate mutated residues. An amino acid insertion and an amino acid deletion are indicated by asterisks. E and F, putative heptad repeat mutants analyzed by SDS-PAGE (E) or native PAGE (F). Lane 1, Gle1152–360; lane 2, Gle1152–360 L248D L251D (2D); lane 3, Gle1152–360 L248D L251D L262D L315D M318D (4D); lane 4, Gle1152–360 L248D L251D L258D L262D L315D M318D (6D); lane 5, Gle1152–360 L248D L251D L258D L262D L315D M318D L322D L325D (8D); lane 6, MBP. G, size-exclusion chromatography analysis of Gle11–360, Gle1152–360, and MBP.
Figure 2
Figure 2
Characterization of an aggregation-prone region of Gle1.A, results of Gle1 sequence submitted to AGGRESCAN server reveals a potential aggregation-prone region. B, size-exclusion chromatography analysis of MBP-Gle11-152, Gle145–54, 152–360, Gle11–360 45–54, and MBP. C, CD spectra of Gle11–360 and the nonvoid volume peak of Gle11–360 45–54. D, disrupting both the coiled-coil domain and the aggregation-prone region of Gle1 impairs oligomerization. Size-exclusion chromatography analysis of Gle11–360, Gle11–360 45–54, and Gle11–3608D is shown.
Figure 3
Figure 3
Gle1 oligomerization required for proper subcellular localization.A, HeLa cells transfected with GFP, GFP-GLE1A, GFP-gle1A-8D, GFP-gle1-ΔA45–54, GFP-gle1A-Δ45–54-8D, GFP-GLE1B, GFP-gle1B-8D, GFP-gle1B-Δ45–54, or GFP-gle1B-Δ45–54-8D were visualized by direct fluorescent live-cell imaging. Scale bar, 10 μm. B, quantification of the GFP nuclear-to-cytoplasmic ratio of HeLa cells expressing GFP-GLE1B, GFP-gle1B-8D, GFP-gle1BΔ-45–54, or GFP-gle1B-Δ45–54-8D either CTRL or GLE1 siRNA-treated. DIC, differential interference contrast.
Figure 4
Figure 4
Gle1B oligomerization mutants have altered nuclear rim localization.A and B, CTRL or GLE1 siRNA-treated HeLa cells were transfected with GFP, GFP-GLE1A, GFP-gle1A-8D, GFP-gle1A-Δ45–54, GFP-gle1A-Δ45–54-8D, GFP-GLE1B, GFP-gle1B-8D, GFP-gle1B-Δ45–54, or GFP-gle1B-Δ45–54-8D plasmids and processed for immunofluorescence using anti-nuclear pore complex proteins antibody (mAb414). The insets highlight nuclear rim with corresponding line intensity plots across the nuclear rim. Scale bar, 10 μm.
Figure 5
Figure 5
mRNA export is disrupted by Gle1B oligomerization mutants.A and B, nuclear poly(A)+ RNA accumulation was assessed in CTRL or GLE1 siRNA-treated HeLa cells transfected with GFP, GFP-GLE1B, GFP-gle1B-8D, GFP-gle1B-Δ45–54, GFP-gle1B-Δ45–54-8D, or GFP-gle1B-Δ45–54-ILLIYSASFLY and detected by in situ Cy3-conjugated oligo(dT) hybridization and direct fluorescence microscopy. Scale bar, 10 μm. C and D, quantification from three independent experiments of the nulclear:cytoplaspmic ratio of poly(A)+ RNA in GFP-positive cells and analyzed using the two-tailed, unpaired t test. ****, p < 0.0001; ***, p = 0.0004. Error bars represent the standard error of the mean.
Figure 6
Figure 6
Gle1A oligomerization mutants do not rescue stress granule defects in the absence of endogenous Gle1 but are phosphorylated.A, HeLa cells treated with either CTRL or GLE1 siRNA were transfected with GFP, GFP-GLE1A, GFP-gle1A-8D, GFP-gle1A-Δ45–54, or GFP-gle1A-Δ45–54-8D, subjected to heat shock at 45 °C for 60 min, and processed for immunofluorescence using anti-G3BP antibody. Scale bar, 10 μm. B and C, analysis of stress granule number (B) and average size (C). Error bars represents the standard error of the mean from three independent experiments. ****, p < 0.0001. D, Gle1A oligomerization mutants are phosphorylated after heat shock treatment. HeLa cells expressing either GFP, GFP-GLE1A, GFP-gle1A-8D, GFP-gle1A-Δ45–54, or GFP-gle1A-Δ45–54-8D in CTRL or GLE1 siRNA-treated cells were subjected to heat shock at 45C for 60 or left untreated. The cell lysates were resolved on SDS-PAGE and immunoblotted with anti-Gle1 antibodies.
Figure 7
Figure 7
Proper Gle1A oligomerization is required for regulating translation.A and B, HeLa cells treated with either CTRL or GLE1 siRNA were transfected with mCherry, mCherry-GLE1A, mCherry-gle1A-8D, mCherry-gle1A-Δ45–54, or mCherry-gle1A-Δ45–54-8D and subjected to heat shock at 45 °C for 60 min (B) or left untreated (A). AHA was added to the incubation for the final 45 min. The samples were processed by Click-iT labeling with Alexa Fluor 488 alkyne for direct fluorescence microscopy. Scale bar, 10 μm. C and D, quantification of mean fluorescence intensity of AHA-484 staining in mCherry-positive cells. Error bars represent the standard error of the mean from three independent experiments.
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