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Dynamic eIF3a O-GlcNAcylation controls translation reinitiation during nutrient stress

Nature Chemical Biology volume 18, pages 134–141 (2022)Cite this article

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Abstract

In eukaryotic cells, many messenger RNAs (mRNAs) possess upstream open reading frames (uORFs) in addition to the main coding region. After uORF translation, the ribosome could either recycle at the stop codon or resume scanning for downstream start codons in a process known as reinitiation. Accumulating evidence suggests that some initiation factors, including eukaryotic initiation factor 3 (eIF3), linger on the early elongating ribosome, forming an eIF3–80S complex. Very little is known about how eIF3 is carried along with the 80S during elongation and whether the eIF3–80S association is subject to regulation. Here, we report that eIF3a undergoes dynamic O-linked N-acetylglucosamine (O-GlcNAc) modification in response to nutrient starvation. Stress-induced de-O-GlcNAcylation promotes eIF3 retention on the elongating ribosome and facilitates activating transcription factor 4 (ATF4) reinitiation. Eliminating the modification site from eIF3a via CRISPR genome editing induces ATF4 reinitiation even under the nutrient-rich condition. Our findings illustrate a mechanism in balancing ribosome recycling and reinitiation, thereby linking the nutrient stress response and translational reprogramming.

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Fig. 1: Prolonged eIF3–80S association in response to amino acid starvation.
Fig. 2: Amino acid starvation triggers de-O-GlcNAcylation of eIF3a.
Fig. 3: Dynamic O-GlcNAcylation of eIF3a regulates translation reinitiation.
Fig. 4: Deficient O-GlcNAcylation of eIF3a promotes eIF3–80S association.
Fig. 5: Deficient O-GlcNAcylation of eIF3a affects general translation reinitiation.
Fig. 6: A model of eIF3–80S association orchestrated by dynamic O-GlcNAcylation of eIF3a.

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Data availability

All sequencing data have been deposited in the Gene Expression Omnibus under accession number GSE181040. Source data are provided with this paper.

Code availability

All the codes used in this study can be found in https://github.com/QianLab-Cornell/Count_Ribo_Reads.git

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Acknowledgements

We thank N. E. Zachara for providing inducible MEF Ogt KO cell lines and R. C. Wek for ATF4 uORF mutants. We also thank Qian lab members for helpful discussions. We are grateful to Cornell University Life Sciences Core Laboratory Center for sequencing support. We thank the Proteomic and MS Facility of Cornell University for help with the mass spectrometry. This work was supported by US National Institutes of Health (R01GM1222814 and DP1GM142101) and HHMI Faculty Scholar (55108556) to S.-B.Q.

Author information

Authors and Affiliations

  1. Division of Nutritional Sciences, Cornell University, Ithaca, NY, USA

    Xin Erica Shu, Yuanhui Mao, Longfei Jia & Shu-Bing Qian

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  1. Xin Erica Shu
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Contributions

S.-B.Q. conceived the project and designed the experiments. X.E.S. designed and performed the majority of experiments. Y.M. conducted the majority of sequencing data analysis. L.J. assisted HiBiT reporter assay and Ribo-seq experiments. All authors discussed the results and edited the manuscript.

Corresponding author

Correspondence to Shu-Bing Qian.

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The authors declare no competing interests.

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Peer review information Nature Chemical Biology thanks Wen Yi and other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Characterization of eIF3-80S association in response to amino acid starvation.

a. MEFs with or without amino acid starvation for 2 hr were subjected polysome profiling using sucrose gradient. Ribosome fractions were collected for immunoblotted using antibodies indicated. The bottom panel shows the eIF3a levels relative to the RpL4. Error bars, mean ± s.e.m.; t-test, two-tailed; n = 2 independent experiments. b. The top panel shows a scatter plot of read density on individual transcripts between 80 S (Ribo-seq, x-axis) and eIF3-80S (eIF3-seq, y-axis) footprints from MEF cells. The bottom panel shows the GO analysis of eIF3-enriched (red) or eIF3-depleted (blue) transcripts. c. The top panel shows a scatter plot of read density on individual transcripts between 80 S (Ribo-seq, x-axis) and eIF3-80S (eIF3-seq, y-axis) footprints from MEF cells subjected to amino acid starvation for 2 hr. The bottom panel shows the GO analysis of eIF3-enriched (red) or eIF3-depleted (blue) transcripts.

Source data

Extended Data Fig. 2 Amino acid starvation induces de-O-GlcNAcylation of eIF3a.

a. Schematic of quantitative mass spectrometry using iTRAQ to compare phosphoproteomics. b. A venn diagram shows the number of proteins identified in control (198), 2 hr amino acid starvation (291), or both (467) from the phosphoproteomics data in (A). c. A scatter plot shows the differential phosphoproteins in MEF cells with and without amino acid starvation. Phosphoproteins with >2 fold increase or decrease are colored in red and blue, respectively. d. MEF/Ogt F/Y(mER-Cre) cells were pre-treated with (+) or without (–) 0.5 μM 4-OHT for 24 hr followed by normal media for another 24 hr. Cells were then subjected to amino acid starvation for 2 hr prior to immunoblotting. Representative results of 3 independent experiments were shown. e. MEF cells as in (D) were subjected to immunoprecipitation using WGA antibody followed by immunoblotting. Representative results of 3 independent experiments were shown. f. HEK293 cells were transfected with scramble or siRNA targeting OGT for 24 hr followed by RL2 (O-GlcNAc) immunoprecipitation and immunoblotting. Representative results of 3 independent experiments were shown. g. MEF cells were treated with increasing doses of Thiamet-G for 16 hr followed by immunoblotting. Representative results of 3 independent experiments were shown.

Source data

Extended Data Fig. 3 Characterization of eIF3-80S association in the presence of Thiamet-G.

a. MEF cells were treated with Thiamet-G for 16 hr with or without amino acid starvation for 2 hr followed by RNA extraction and RT-qPCR. Relative Atf4 mRNA levels are normalized to the Actb mRNA level. Error bars, mean ± s.d.; n = 3 independent experiments. b. MEF cells were treated with 0.4 μM Thiamet-G for 1 hr followed by amino acid starvation for 2 hr. Whole-cell lysates were immunoprecipitated using the eIF3a antibody followed by immunoblotting using indicated antibodies. Representative results of 3 independent experiments were shown. c. Same as (b), total RNAs and eIF3-associated RNAs were extracted followed by RT-PCR measuring Actb and Aft4 mRNA levels. Relative Atf4/Actb ratios were normalized to the corresponding input. Error bars, mean ± s.d.; *p = 0.019; ***p = 0.00078; two-way ANOVA; n = 3 independent experiments. d. MEF cells as in (b) were subjected to immunoblotting using antibodies indicated. The right panel shows the relative ATF4 protein levels normalized to α-tubulin. Error bars, mean ± s.d.; ***p = 0.00014; two-way ANOVA; n = 3 independent experiments. e. MEF cells were treated with Thiamet-G for 16 hr were subjected to polysome profiling using sucrose gradient. Ribosome fractions were collected for immunoblotted using antibodies indicated. The bottom panel shows the densitometry of eIF3a and RpL4 protein levels. Representative results of 3 independent experiments were shown. f. MEF cells as (e) were subjected to amino acid starvation for 2 h followed by polysome profiling using sucrose gradient. Ribosome fractions were collected for immunoblotted using antibodies indicated. The bottom panel shows the densitometry of eIF3a and RpL4 protein levels. Representative results of 3 independent experiments were shown.

Source data

Extended Data Fig. 4 Further characterization of eIF3-80S association in the presence of Thiamet-G.

a. MEF cells were treated with Thiamet-G for 16 hr followed by amino acid starvation for 2 hr. Polysome profiling was conducted using sucrose gradient. Ribosome fractions were collected for immunoblotted using antibodies indicated. Representative results of 3 independent experiments were shown. b. The top panel shows a scatter plot of read density on individual transcripts between 80 S (Ribo-seq, x-axis) and eIF3-80S (eIF3-seq, y-axis) footprints from MEF cells treated with Thiamet-G. The bottom panel shows the GO analysis of eIF3-enriched (red) or eIF3-depleted (blue) transcripts. c. The top panel shows a scatter plot of read density on individual transcripts between 80 S (Ribo-seq, x-axis) and eIF3-80S (eIF3-seq, y-axis) footprints from MEF cells treated with Thiamet-G and 2 hr amino acid starvation. The bottom panel shows the GO analysis of eIF3-enriched (red) or eIF3-depleted (blue) transcripts. d. The distribution of 80 S footprints in the Atf4 transcript from MEF cells with or without Thiamet-G treatment, before and after amino acid starvation.

Source data

Extended Data Fig. 5 Deficient O-GlcNAcylation attenuates global protein synthesis.

a. MEF/Ogt F/Y(mER-Cre) cells were pre-treated with (+) or without (–) 0.5 μM 4-OHT for 24 hr followed by normal media for another 24 hr. Cells were then subjected to amino acid starvation for 2 hr followed by immunoprecipitation using the eIF3a antibody. Total RNAs and eIF3-associated RNAs were extracted followed by RT-PCR measuring Actb and Aft4 mRNA levels. Relative Atf4/Actb ratios were normalized to the corresponding input. Error bars, mean ± s.e.m.; *P = 0.0036; two-way ANOVA; n = 3 independent experiments. b. MEF cells as in (a) were subjected to immunoblotting using antibodies indicated. The right panel shows the relative ATF4 protein levels normalized to α-tubulin. Error bars, mean ± s.e.m.; *P = 0.015, **P = 0.0067; two-way ANOVA; n = 3 independent experiments. c. MEF cells as in (a) were subjected to polysome profiling using sucrose gradient. Ribosome fractions were collected for immunoblotted using antibodies indicated. The bottom panel shows the eIF3a levels relative to the RpL4. Error bars, mean ± s.e.m.; t-test, two-tailed; n = 2 independent experiments. The bottom panel shows the densitometry of eIF3a and RpL4 protein levels. d. MEF cells as in (a) were incubated with 200 ng/µl of puromycin for 15 min. Whole cell lysates were used for immunoblotting. Representative results of 3 independent experiments were shown. e. MEF cells as in (a) were subjected to polysome profiling using sucrose gradient. Ribosome fractions were collected for immunoblotted using antibodies indicated. Representative results of 3 independent experiments were shown.

Source data

Extended Data Fig. 6 Deficient O-GlcNAcylation of eIF3a promotes eIF3-80S association.

a. The top panel shows a scatter plot of read density on individual transcripts between 80 S (Ribo-seq, x-axis) and eIF3-80S (eIF3-seq, y-axis) footprints from MEF cells with 4-OHT treatment. The bottom panel shows the GO analysis of eIF3-enriched (red) or eIF3-depleted (blue) transcripts. b. The top panel shows a scatter plot of read density on individual transcripts between 80 S (Ribo-seq, x-axis) and eIF3-80S (eIF3-seq, y-axis) footprints from MEF cells with 4-OHT treatment and 2 hr amino acid starvation. The bottom panel shows the GO analysis of eIF3-enriched (red) or eIF3-depleted (blue) transcripts. c. A Scatter plot shows the correlation of fold changes (log2) of CDS ribosome density in MEF/Ogt F/Y(mER-Cre) cells between Ogt knockout and amino acid starvation. d. Cumulative distribution of fold changes of read density in 5’UTR (blue) and 3’UTR (red) relative to the CDS in MEF cells with OGT knockout (top panel) or together with amino acid starvation (bottom panel). P values are based on Wilcoxon signed-rank test.

Extended Data Fig. 7 Characterization of eIF3a mutants with deficient O-GlcNAcylation.

a. HEK 293 cells were transfected with plasmids expressing EGFP-eIF3a for 24 hr. Cells lysates were immunoprecipitated using an anti-GFP antibody followed by immunoblotting. Bottom panel, HEK cells transfected with GFP-eIF3a were subjected to a 15%-45% sucrose gradient, followed by western blotting using indicated antibodies. Representative results of 3 independent experiments were shown. b. HEK 293 cells were transfected with plasmids expressing wild type eIF3a (WT) or T336A mutant fused to EGFP for 24 hr. Cells lysates were immunoprecipitated using an anti-GFP antibody followed by immunoblotting. Representative results of 3 independent experiments were shown. c. HEK 293 cells bearing eIF3a wildtype (WT) and S225G mutation were incubated with 200 ng/µl of puromycin for 15 min. Whole cell lysates were used for immunoblotting. d. Growth rates of HEK 293 cells bearing eIF3a wildtype (WT) and S225G mutation were measured using CCK-8 assay. Error bars, mean ± s.d.; t-test, two-tailed; n = 3 biological replicates. e. HEK293 cells as in (C) were subjected to amino acid starvation for 2 hr followed by total RNA extraction and RT-qPCR. Relative Atf4 mRNA levels are normalized to the Actb mRNA level. Error bars, mean ± s.d.; n = 3 independent experiments.

Source data

Extended Data Fig. 8 Deficient O-GlcNAcylation of eIF3a affects general translation reinitiation.

a. The left panel shows the schematic of ATF4-Fluc reporters with uORF deletion. The right panel shows the Fluc activities in transfected MEF cells with and without amino acid starvation. Error bars, mean ± s.d.; *p = 0.037; two-way ANOVA; n = 3 independent experiments. b. MEF cells transfected with ATF4-Fluc reporter mutants were subjected to amino acid starvation for 2 hr. Whole cell lysates were immunoprecipitated using the eIF3a antibody. Total RNAs and eIF3-associated RNAs were extracted followed by RT-PCR measuring Actb and Aft4 mRNA levels. Relative Atf4/Actb ratios were normalized to the corresponding input. Error bars, mean ± s.d.; ***p = 0.00035; two-way ANOVA; n = 3 independent experiments. c. HEK293 cells bearing S225G mutation of eIF3a were transfected with ATF4-Fluc reporter mutants followed by amino acid starvation for 2 hr. Whole cell lysates were immunoprecipitated using the eIF3a antibody. Total RNAs and eIF3-associated RNAs were extracted followed by RT-PCR measuring Actb and Aft4 mRNA levels. Relative Atf4/Actb ratios were normalized to the corresponding input. Error bars, mean ± s.d.; n = 3 independent experiments. d. EK293 cells with (+) or without (–) amino acid starvation for 2 hr were subjected to immuno-precipitation using eIF3a antibody. Total RNAs and eIF3-associated RNAs were extracted followed by RT-PCR measuring Actb and Map2k6 mRNA levels. Relative Map2k6/Actb ratios were normalized to the corresponding input. Error bars, mean ± s.d.; *p = 0.018; two-way ANOVA; n = 3 independent experiments. e. Steady-state mRNA levels of Map2k6 in HEK293 cells as in (d) were normalized with Actb, Error bars, mean ± s.d.; n = 3 independent experiments.

Source data

Extended Data Fig. 9 A model summarizing eIF3-80S complex association orchestrated by dynamic O-GlcNAcylation of eIF3a.

Schematic Cryo-EM structure of mammalian eIF3 (Adopted from des Georges et al. 2015). eIF3a is shown in red and the putative O-GlcNAc modification site shown as the white star.

Supplementary information

Reporting Summary

Supplementary Table 1

Phosphoproteomics of MEF cells with or without amino acid starvation.

Supplementary Table 2

Quantitative proteomics of O-GlcNAc modification in MEF cells with or without amino acid starvation.

Supplementary Table 3

Quantitative proteomics of O-GlcNAc modification in MEF cells with or without OGT knockdown.

Supplementary Table 4

Tandem mass spectrometry of purified eIF3a for putative O-GlcNAc modification sites.

Source data

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Shu, X.E., Mao, Y., Jia, L. et al. Dynamic eIF3a O-GlcNAcylation controls translation reinitiation during nutrient stress. Nat Chem Biol 18, 134–141 (2022). https://doi.org/10.1038/s41589-021-00913-4

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