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. 2017 Oct 27;18(1):201.
doi: 10.1186/s13059-017-1338-4.

Dynamic changes in eIF4F-mRNA interactions revealed by global analyses of environmental stress responses

Joseph L Costello  1   2 , Christopher J Kershaw  1 , Lydia M Castelli  1   3 , David Talavera  4 , William Rowe  1   5 , Paul F G Sims  6 , Mark P Ashe  1 , Christopher M Grant  1 , Simon J Hubbard  7 , Graham D Pavitt  8
Affiliations

Dynamic changes in eIF4F-mRNA interactions revealed by global analyses of environmental stress responses

Joseph L Costello et al. Genome Biol. .

Abstract

Background: Translation factors eIF4E and eIF4G form eIF4F, which interacts with the messenger RNA (mRNA) 5' cap to promote ribosome recruitment and translation initiation. Variations in the association of eIF4F with individual mRNAs likely contribute to differences in translation initiation frequencies between mRNAs. As translation initiation is globally reprogrammed by environmental stresses, we were interested in determining whether eIF4F interactions with individual mRNAs are reprogrammed and how this may contribute to global environmental stress responses.

Results: Using a tagged-factor protein capture and RNA-sequencing (RNA-seq) approach, we have assessed how mRNA associations with eIF4E, eIF4G1 and eIF4G2 change globally in response to three defined stresses that each cause a rapid attenuation of protein synthesis: oxidative stress induced by hydrogen peroxide and nutrient stresses caused by amino acid or glucose withdrawal. We find that acute stress leads to dynamic and unexpected changes in eIF4F-mRNA interactions that are shared among each factor and across the stresses imposed. eIF4F-mRNA interactions stabilised by stress are predominantly associated with translational repression, while more actively initiating mRNAs become relatively depleted for eIF4F. Simultaneously, other mRNAs are insulated from these stress-induced changes in eIF4F association.

Conclusion: Dynamic eIF4F-mRNA interaction changes are part of a coordinated early translational control response shared across environmental stresses. Our data are compatible with a model where multiple mRNA closed-loop complexes form with differing stability. Hence, unexpectedly, in the absence of other stabilising factors, rapid translation initiation on mRNAs correlates with less stable eIF4F interactions.

Keywords: Stress regulation of gene expression; Translational control; Yeast; eIF4F.

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The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Stress treatment does not affect ‘closed-loop’ factor associations. a Polysome profiles of each tagged strain before and following acute stress are unaffected by the TAP tag. b TAP-IP recovers equivalent levels of associated factors ± stress. Minor variations seen are not reproducible. c Overview of approach to generate samples for RNA sequencing
Fig. 2
Fig. 2
Overall changes in mRNA association with eIF4F following different stresses (∆IP). Pairwise scatterplots showing differential association of 5348 mRNAs with each eIF4F factor following stress (log2 fold change stress/unstressed: ∆IP). mRNAs where association changes the most following stress are located at the extremes. Top: eIF4E plots; middle: eIF4G1 plots; bottom: eIF4G2 plots. In each row: left: ± aa (x-axis) plotted against ± glu (y-axis); middle: ± aa (x-axis) against ± H2O2 (y-axis); right: ± glu (x-axis) against ± H2O2 (y-axis)
Fig. 3
Fig. 3
Reciprocal changes in transcription (∆T) and eIF4F association (∆IP) in response to stress. a Box-and-whisker plots showing the distribution of changes in IP (∆IP) with eIF4F proteins for mRNAs transcriptionally upregulated (up, red) downregulated (dn, blue) or statistically (FDR < 0.05) not changed (nc, gold, green or light blue) in response to the same stress in the same strain. b Scatter plots of 5348 mRNAs showing change in transcription (∆T) and change in eIF4F association with stress (∆IP). mRNAs whose factor association changes significantly (FDR < 0.05) following stress are highlighted in red (up) and blue (down), based on edgeR analyses, see Additional file 5: Supplementary Source Data 4
Fig. 4
Fig. 4
Reciprocal changes in relative ribosome occupancy –translation efficiency (∆TE) and eIF4F association (∆IP) in response to stress. a Box-and-whisker plots showing the distribution of changes in TE with eIF4F factor association. mRNAs statistically (FDR < 0.05) enriched in IP following stress (up, red) depleted (dn, blue) or not changed (nc, gold, green or blue) in response to the same stress in the same strain. ∆TE calculated from previously published experiments. b Scatter plots of the same mRNAs highlighting specific labelled mRNAs discussed in the text
Fig. 5
Fig. 5
Opposing responses to stress of ‘closed-loop’ Group I and IVA mRNAs. a Left: median log2 fold change for Group I–IVC mRNAs as defined by Costello et al. [13]. See Additional file 2: Figure S6A for box plot representations of this data. ∆IP change in eIF4F association (middle) and ∆TE (right) are shown for each group across each of the three stresses. Colour key in box. b, c Box plots showing the effect of change in eIF4E association with each stress (b) and ∆TE (c) on each gene cluster ∆IP denoted by a specific colour (G I red; G II blue; G IIIA and B shades of green; G IVA-C shades of purple). d Pairwise plots showing changes in IP and TE for G I and G IVA for eIF4E. Specific mRNAs are indicated with arrows. mRNA groups are denoted by a specific colour (G I red; G II blue; G IIIA and B shades of green; G IVA-C shades of purple). b–d Top ± amino acids plots, middle ± H2O2, bottom ± glucose. Equivalent plots to panels (b) and (d) for eIF4G1 and eIF4G2, respectively, are shown in Additional file 2: Figure S6B and C
Fig. 6
Fig. 6
Calculated translation initiation times anti-correlate with eIF4F association in unstressed cells. a No correlation between length of 5′ UTR and initiation time calculated from ribosome profiling experiments in unstressed cells [12]. b Anti-correlation between eIF4F factor association and calculated initiation time. c Mean calculated initiation time across Costello et al. [13] mRNA Groups I–IVC
Fig. 7
Fig. 7
Model for differential eIF4F associations during translation initiation. Diagram depicts three different states of closed-loop mRNA complex. Bottom: State 1: a non-initiating mRNA. Top left: State 2: a 48S bound complex with contacts between eIF4G and recruited 43S factors during which scanning occurs. Top right: State 3: a post-AUG recognition complex undergoing 60S joining where initiation factors including eIF2-GDP are released. Actively initiating mRNAs likely cycle between the conformations 2 and 3 (grey arrows), correlating with low eIF4F affinity or recovery. mRNAs initiating rapidly under optimal growth conditions, but sensitive to stress transition to a conformation where eIF4F becomes more stably associated and TE lowered: red arrow to state 1. Other mRNAs have high affinity for 4 F and are initiated less frequently (state 1), but can be activated to recruit more ribosomes following stress, entering a dynamic state where 4 F affinity is lowered: green arrow to state 2
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