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. 2010 Apr 1;5(4):e9942.
doi: 10.1371/journal.pone.0009942.

eIF5A promotes translation elongation, polysome disassembly and stress granule assembly

Chi Ho Li  1 , Takbum Ohn, Pavel Ivanov, Sarah Tisdale, Paul Anderson
Affiliations

eIF5A promotes translation elongation, polysome disassembly and stress granule assembly

Chi Ho Li et al. PLoS One. .

Abstract

Stress granules (SGs) are cytoplasmic foci at which untranslated mRNAs accumulate in cells exposed to environmental stress. We have identified ornithine decarboxylase (ODC), an enzyme required for polyamine synthesis, and eIF5A, a polyamine (hypusine)-modified translation factor, as proteins required for arsenite-induced SG assembly. Knockdown of deoxyhypusine synthase (DHS) or treatment with a deoxyhypusine synthase inhibitor (GC7) prevents hypusine modification of eIF5A as well as arsenite-induced polysome disassembly and stress granule assembly. Time-course analysis reveals that this is due to a slowing of stress-induced ribosome run-off in cells lacking hypusine-eIF5A. Whereas eIF5A only marginally affects protein synthesis under normal conditions, it is required for the rapid onset of stress-induced translational repression. Our results reveal that hypusine-eIF5A-facilitated translation elongation promotes arsenite-induced polysome disassembly and stress granule assembly in cells subjected to adverse environmental conditions.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Depletion of ornithine decarboxylase (ODC) inhibits SG assembly.
(A) RDG3 (GFP-G3BP;RFP-Dcp1a) double stable cells were transfected with control (siCONT) or ODC-specific (siODC1) siRNAs, then cultured in the absence (Mock) or presence (Arsenite) of 120 µM sodium arsente for 30 min before processing for immunofluorescence microscopy. G3BP-GFP was used to visualize SGs (left panels), DCP1a-RFP was used to visualize PBs (right panels). Scale bar, 10 µm. (B) U2OS cells were transfected with control (siCONT) or ODC-specific (siODC1) siRNAs, then cultured in the presence of 120 µM arsenite for 30 min before processing for immunofluorescence microscopy using antibodies reactive with eIF3b (SG marker) and Rck (PB marker). Scale bar, 10 µm. (C) Knockdown efficiency of ODC1 mRNA was assessed using RT-qPCR in both RDG3 and U2OS cells. Results are reported as means ± S.D., n = 4. (D) Graphical representation of SG assembly in RDG3 and U2OS cells transfected with control (siCONT) or non-overlapping ODC1-specific (siODC1-1 and siODC1-2) siRNAs prior to treatment with 120 µM arsenite for 30 min. Cell counting data are shown as means ± S.D. from different experiments (siODC1-1, n = 2; siODC1-2, n = 1 for each cell line).
Figure 2
Figure 2. Depletion of eIF5A inhibits SG assembly.
(A) RDG3 cells were transfected with control (siCONT) or eIF5A-specific (siEIF5A-1 and siEIF5A-2) siRNAs, then cultured in the absence (Mock) or presence (Arsenite) of 120 µM arsenite for 30 min before processing for immunofluorescence microscopy. G3BP-GFP was used to visualize SGs (left panels), DCP1a-RFP was used to visualize PBs (right panels). Scale bar, 10 µm. (B) U2OS cells were transfected with control (siCONT) or eIF5A-specific (siEIF5A) siRNAs, then cultured in the absence (Mock) or presence (Arsenite) of 120 µM arsenite for 30 min before processing for immunofluorescence microscopy using antibodies reactive with eIF3b (SG marker) and Rck (PB marker). Scale bar, 10 µm. (C) Western blot analysis quantifying the knockdown efficiency of eIF5A in both U2OS and RDG3 cells (upper panel). Actin was used as loading control (lower panel). (D) Graphical representation of SG assembly in RDG3 and U2OS cells transfected with control (siCONT) or non-overlapping EIF5A-specific (siEIF5A-1 and siEIF5A-2) siRNAs prior to treatment with 120 µM arsenite for 30 min. Cell counting data are shown as means ± S.D. from different experiments. *, P<0.01 (siEIF5A-1, n = 4 (RDG3) and n = 3 (U2OS); siEIF5A-2, n = 2 for each cell line).
Figure 3
Figure 3. eIF5A is concentrated at SGs.
COS7 cells were treated with sodium arsenite (0.5 mM), clotrimazole (20 µM) or heat shock (44°C) for 1 hour prior to processing for immunofluorescence microscopy using antibodies reactive with eIF3b (green, SG marker), Rck (blue, PB marker), and eIF5A (red). Enlarged views of boxed areas show individual channels and merged views.
Figure 4
Figure 4. Hypusination of eIF5A is required for SG assembly.
(A) U2OS cells transfected with control (siCONT) or DHS-specific (siDHS-1 and siDHS-2) siRNAs were cultured in the absence (Mock) or presence (Arsenite) of 120 µM arsenite for 30 min before processing for immunofluorescence microscopy using antibodies reactive with eIF3b (SG marker) and Rck (PB marker). Scale bar, 10 µm. (B) Hypusination inhibitor GC7 inhibits SG assembly. U2OS cells were treated with GC7 (10 µM) or vehicle control (Mock) for 48 hours prior to culture in the absence (Untreat) or presence (Arsenite) of 0.1 mM sodium arsenite for 1 hour, then processed for immunofluorescence microscopy using antibodies reactive with Hedls (PB marker) and eIF3b (SG marker). Merged views are shown in the right panels. Scale bar, 10 µm. (C) Western blot analysis quantifying the knockdown efficiency of DHS (upper panel). Actin was used as loading control (lower panel). (D) Graphical representation of SG assembly in U2OS cells transfected with control (siCONT) or non-overlapping DHS-specific (siDHS-1 and siDHS-2) siRNAs prior to treatment with 120 µM arsenite for 30 min. Cell counting data are shown as means ± S.D. from different experiments.*, p<0.05; **, p<0.01 (siDHS-1, n = 3; siDHS-2, n = 2). (E) U2OS cells cultured with vehicle (Mock) or GC7 (10 µM) as in (B). The percentage of cells with SGs was quantified microscopically and graphed as an average ± S.D. (n = 2).
Figure 5
Figure 5. Isoelectric focusing analysis quantifies eIF5A hypusination.
U2OS cells were transfected with control (CONT), DHS-specific (DHS), or ODC1-specific (ODC1) siRNAs or treated with GC7 (10 µM) for 1 hour prior to processing for IEF analysis. U2OS cells expressing recombinant eIF5A (K50A) was used as a reference standard for unhypusinated eIF5A. The percentage of unhypusinated eIF5A in each sample was quantified using densitometric analysis.
Figure 6
Figure 6. Knockdown of ODC1 or eIF5A inhibits stress-induced polysome disassembly.
(A) U2OS cells were transfected with control (CONT KD), ODC1-specific (ODC1KD) or eIF5A-specific (EIF5A KD) siRNAs, then cultured in the absence (−ars) or presence (+ars) of 0.1 mM sodium arsenite before processing for sucrose gradient analysis. The migration of 40S, 60S and 80S ribosomes is indicated by arrows. The migration of preserved low density polysomes is indicated by brackets. (B) The analysis described in (A) was repeated using prolonged centrifugation times and more sensitive readings to improve the resolution of ribosomal subunits. (C) Fractions from control and eIF5A knockdown sucrose gradients were analyzed by Western blotting using antibodies reactive with RPS3 and RPL13a.
Figure 7
Figure 7. GC7-mediated hypusination inhibition phenocopies eIF5A knockdown.
U2OS cells were treated with GC7 (10 µM) or vehicle control (Mock) for the indicated times before culturing cells in the absence (−ars) or presence (+ars) of 0.1 mM sodium arsenite for 1 hour and processing for sucrose gradient analysis. The migration of 60S ribosomal subunits and preserved low density polysomes are indicated.
Figure 8
Figure 8. Hypusine-modified eIF5A is required for stress-induced translational arrest.
(A) U2OS cells transfected with control (siCONT), DHS-specific (siDHS), or ODC1-specific (siODC1) siRNAs were cultured in the absence or presence of 0.1 mM sodium arsenite for 30 minutes, then pulsed with 35S-methionine labeling medium for an additional 30 minutes. Cells were washed and acetone precipitated to quantify 35S-methionine incorporation. The ratio of 35S-methionine incorporation (CPM/µg protein) in arsenite treated (ARS) vs. untreated cells is reported as mean ± (S.E) from the indicated number of experiments. (B) SDS-PAGE analysis of metabolically labeled protein in cell extracts with or without sodium arsenite treatment. Equivalent amounts of total protein were loaded in each lane.
Figure 9
Figure 9. Depletion of eIF5A delays stress-induced polysome disassembly.
(A) Polysome profiling analysis in U2OS cells treated with a control siRNA. Upper panels show polysome profiles obtained from a UV monitor (254 nm). Peaks corresponding to 40S, 60S and 80S ribosomes as well as polysomes are indicated with arrows. Middle panels show western blots of individual fractions across the gradient probed with antibodies against RPS3 and RPL13a. Bottom panels are graphical representations of quantitative distribution analyses of RPS3 and RPL13a in polysome fractions 6–10. The abundance of ribosomal proteins in each fraction was divided by the total abundance across the polysome fractions. (B) Polysome profiling analysis in U2OS cells treated with eIF5A-specific siRNAs. Panel descriptions are as in (A). (C) Pulse labeling with 35S-methionine. U2OS cells were treated with the indicated siRNAs, cultured in the absence or presence of arsenite, and pulsed with 35S-methionine prior to SDS-PAGE and autoradiographic analysis. In cells labeled “pulse 30”, 35S-methionine was added 30 minutes after the addition of arsenite. In cell labeled “pulse 60”, 35S-methionine was added 60 minutes after the addition of arsenite. (D) Logarithm of the ratio of 35S incorporation (CPM/µg) in arsenite/untreated U2OS cells after the pulse 30 or pulse 60 (n = 3, Error bars denote ± S.D).
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