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. 2013 Apr 17;32(8):1087-102.
doi: 10.1038/emboj.2013.61. Epub 2013 Mar 22.

TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h

Affiliations

TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h

Mikhail Schepetilnikov et al. EMBO J. .

Abstract

Mammalian target-of-rapamycin (mTOR) triggers S6 kinase (S6K) activation to phosphorylate targets linked to translation in response to energy, nutrients, and hormones. Pathways of TOR activation in plants remain unknown. Here, we uncover the role of the phytohormone auxin in TOR signalling activation and reinitiation after upstream open reading frame (uORF) translation, which in plants is dependent on translation initiation factor eIF3h. We show that auxin triggers TOR activation followed by S6K1 phosphorylation at T449 and efficient loading of uORF-mRNAs onto polysomes in a manner sensitive to the TOR inhibitor Torin-1. Torin-1 mediates recruitment of inactive S6K1 to polysomes, while auxin triggers S6K1 dissociation and recruitment of activated TOR instead. A putative target of TOR/S6K1-eIF3h-is phosphorylated and detected in polysomes in response to auxin. In TOR-deficient plants, polysomes were prebound by inactive S6K1, and loading of uORF-mRNAs and eIF3h was impaired. Transient expression of eIF3h-S178D in plant protoplasts specifically upregulates uORF-mRNA translation. We propose that TOR functions in polysomes to maintain the active S6K1 (and thus eIF3h) phosphorylation status that is critical for translation reinitiation.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Auxin-induced phosphorylation of AtS6K1 at TOR-specific residue T449 regulates interaction with eIF3. (A) eIF3c or TOR immunoprecipitated from WT extracts was assayed for complex formation between eIF3c, TOR, S6K1, raptor, and ARF-GTPase. Input, 5% of immunoprecipitation (IP) or normal rabbit serum (RS). (B) Suspension-culture cells treated with NAA or Torin-1 for 0, 4, and 6 h, lysed assayed by immunoblot analysis. (C) eIF3c immunoprecipitated from extracts in (B) and assayed for association with TOR and S6K1 by immunoblotting. Source data for this figure is available on the online supplementary information page.
Figure 2
Figure 2
uORF-mRNA abundance in polysomes is regulated by auxin and Torin-1. (A) Schematic representation of mRNAs coding for ARFs, bZIP11, IAA6, and Actin (open rectangles uORFs). (B) Distribution of mRNAs—ARF3, ARF5, ARF6, ARF11, bZIP11, IAA6 and Actin analysed in polysome gradient fractions from extracts prepared from 7-dag seedlings treated (or not, Mock) with either auxin (NAA) or Torin-1 for 8 h. A set of graphs shows quantification of semi-quantitative RT–PCR (sqRT–PCR; Supplementary Figure S2A) corrected for polysome volume in non-, or NAA-, or Torin-1-treated ribosomal profiles. The highest value of each WT/NAA polysome-bound mRNA was set as 100%. Error bars indicate standard deviation of the mean of three replicates. (C) Quantitative RT–PCR (qRT–PCR) of each mRNA in total extracts as in (B). The RNA value in WT/Torin-1 extracts was set as 100%. Values, expressed in arbitrary units, are averages of two replicates, and error bars indicate s.d. (D) Alignment of phosphorylation site patterns from TOR homologues Human, Rattus norvegicus, Arabidopsis, Zea mays, and Oryza sativa. The human phosphorylation site S2448 within the motif is indicated. Similar residues are printed in reverse type and conserved residues are shaded in agreement with Blossom 62 and Jonson amino-acid substitution matrixes. (E) Time course of ARF5::GFP accumulation in 7-dag seedlings expressing GFP tag fused to ARF5 under the control of the natural promoter (ARF5:ARF5::GFP) before (0 h) and after transfer to medium with NAA or Torin-1 analysed by immunoblot with anti GFP ABs (see quantification below the blot line). TOR-P, TOR, and eIF3c protein values in above conditions assayed by immunoblot and ARF5::GFP were corrected for loading control (LC; TOR phosphorylation was quantified). The value at 0 h (no incubation) for each line was set as 100%. Data shown are the means of three independent blots. Source data for this figure is available on the online supplementary information page.
Figure 3
Figure 3
TOR and S6K1 are loaded on polysomes in an NAA- and Torin-1-sensitive manner. (AC) Ribosomal profiles obtained from extracts prepared from 7-dag seedlings treated (or not, A) with either auxin (NAA, B) or Torin-1 (C) for 8 h. In all, 1 ml (1V, 80S/60S/40S) and 2 ml (2V, polysomes) aliquots were precipitated with 10% TCA; rRNA was analysed by agarose gel electrophoresis, and S6K1/TOR by immunoblot. Left (2V) and right (1V) panels below each profile are images from the same gel. Data shown are representative of three independent blots. (D, E) Ribosomal profiles of polyribosomes and ribosomal species from extracts prepared from auxin treated with RNase A (D) and Torin-1 treated with RNase A (E). Source data for this figure is available on the online supplementary information page.
Figure 4
Figure 4
Polysomal loading of both uORF-mRNAs and TOR is impaired in TOR RNAi plants. (A) Comparison of uORF-RNA accumulation in polysomes in extracts prepared from 7-dag TOR RNAi seedlings treated (or not, Mock) with NAA or Torin-1 for 8 h. A set of graphs shows quantification of sqRT–PCR (Supplementary Figure S4A) corrected for polysome volume in non-, or NAA-, or Torin-1-treated ribosomal profiles. The highest value of each mRNA in polysomes from NAA/WT plants (Figure 2B) was set as 100%. Error bars indicate s.d. of the mean of three replicates. (B) qRT–PCR of mRNAs in total extracts prepared as in (A). The RNA value in TOR RNAi/Torin-1 extracts was set as 100%. Values are averages of three replicates. (C) Immunoblot analysis of TOR, S6K1, and eIF3h phosphorylation, as well as their accumulation levels in the control line (WT) and the TOR-deficient RNAi line. LC, loading control. (D) Ribosomal profiles from TOR RNAi were obtained as in (B). In all, 2 ml (2V, polysomes) and 1 ml (1V, 80S/60S/40S) were used to monitor distribution of rRNA on agarose gels and S6K1/TOR by immunoblot. Left (2V) and right (1V) panels of rRNA gel and immunoblot are from the same gel. Source data for this figure is available on the online supplementary information page.
Figure 5
Figure 5
Polysomal loading of uORF-mRNAs, but not TOR signalling, is impaired in eif3h-1 plants. (A) Comparison of uORF-RNA accumulation in polysomes in extracts prepared from 7-dag eif3h-1 seedlings treated for 8 h with NAA or Torin-1. Quantification of sqRT–PCR data (Supplementary Figure S5A) corrected for polysome volume in NAA- or Torin-1-treated ribosomal profiles. The highest value of each mRNA in NAA/WT polysomes (Figure 2B) was set as 100%. Error bars indicate s.d. of the mean of two replicates. (B) qRT–PCR of mRNAs in total eif3h-1 extracts prepared as in (A). The RNA value in eif3h-1/Torin-1 extracts was set as 100%. Each value is the average of two replicates. (C) Immunoblot analysis of TOR, S6K1, and eIF3h phosphorylation, as well as their accumulation levels in WT and the eif3h-1 line. LC, loading control. (D) Ribosomal profiles from eif3h-1 obtained as in (A). In all, 2 ml (2V, polysomes) and 1 ml (1V, 80S/60S/40S) were used to monitor rRNA and S6K1/TOR distribution by immunoblot. Left (2V) and right (1V) panels of rRNA gel and immunoblot are from the same gel. Source data for this figure is available on the online supplementary information page.
Figure 6
Figure 6
Auxin triggers eIF3h phosphorylation and association with polysomes in a Torin-1-sensitive manner. (A) Alignment of phosphorylation site patterns from eIF3h homologues Mus musculus, Homo sapiens, Arabidopsis, Oryza sativa, Zea mays, Medicago truncatula, and Vitis vinifera. Akt/S6K1 phosphorylation site consensus (R/KxR/KxxS/T) within the motif is underlined (putative phosphorylation residue in red). (B) The putative AteIF3h 3D structure was generated by PyMOL. The S178-P position within the conserved loop shown in (A) is highlighted in red, residues predicted to fold as helixes in green and β-sheets in yellow. (C) WT/NAA and WT/Torin-1 protein extracts were resolved in two dimensions and revealed by western blot with anti-eIF3h or anti-(R/KxR/KxxS/T-P) antibodies. Molecular masses are indicated. Arrow, eIF3h-P. (D–G) Ribosomal profiles from WT (D) not treated or WT, TOR RNAi, eif3h-1 treated with NAA or Torin-1 (EG, respectively) were obtained as in (Figure 3) and indicated by distribution of rRNA. In all, 2 ml (2V, polysomes) and 1 ml (1V, 80S/60S/40S) were used to monitor distribution of rRNA by agarose gel and eIF3h/eIF3h-P by immunoblot with antibodies as in (C). Left (2V) and right (1V) panels of rRNA gel and immunoblot are from the same gel. (H) Extracts prepared from WT seedlings were used for immunoblotting of eIF3h, TOR, or S6K1 present in Input, normal rabbit serum (RS), and the entire immunoprecipitate (IP). Source data for this figure is available on the online supplementary information page.
Figure 7
Figure 7
Auxin and Torin-1 regulate reinitiation after uORF translation in Arabidopsis suspension protoplasts. (A) Transient expression experiments in Arabidopsis suspension protoplasts included the two reporter plasmids (left panel): pmonoGFP and pmonoGUS; and pmonoGFP and either pMAGRIS-GUS, or pMAGDIS-GUS in the amounts indicated below the graphs (right panel). After transfection, cells were incubated with Torin-1 or not (Mock) for 18 h, and GUS/GFP ratios are shown as open (pmonoGUS/pmonoGFP) and black (pMAGRIS/MAGDIS-GUS/pmonoGFP) bars. pmonoGUS expression in Mock protoplasts was set as 100% (197 800 RFU). Reporter mRNA levels were analysed by sqRT–PCR. (B) Arabidopsis suspension protoplasts were transformed with pmonoGFP and either pmonoGUS or pARF5-GUS (left panel). After transfection, cells were incubated or not (Mock) with Torin-1 or NAA or (NAA+Torin-1) for 18 h, and GUS/GFP ratios were calculated and shown as open (for pmonoGUS/pmonoGFP) and black bars (for pARF5-GUS/pmonoGFP). The GUS/GFP ratio found in Mock protoplasts (for pmonoGUS/pmonoGFP) was set as 100% (211 000 RFU). TOR, S6K1, and their phosphorylation status in protoplasts were assayed by immunoblotting (left panel). Densitometry was used to quantify western blot results from at least three independent replicates (NAA value set as 100%). (C) pmonoGFP and either pARF3-GUS or pARF3Δ(AUG1+2)-GUS or pARF3ΔAUG1-GUS or pARF3ΔAUG2-GUS (left panel) were used for transformation. GUS/GFP ratios were calculated and shown as open (Mock) and black bars (Torin-1). The GUS/GFP ratio found in Mock protoplasts with uORF-less ARF3 leader was set as 100% (150 000 RFU). (D) Protoplasts transformation with pmonoGFP and either pARF3Δ(AUG1+2)-GUS (open bars) or pARF3-GUS (black bars) with or without additional plasmids indicated below the graphs. The GUS/GFP ratio found in Mock protoplasts with uORF-less ARF3 leader was set as 100% (180 000 RFU). eIF3h, S6K1, and their phosphorylation status in protoplasts were assayed by immunoblotting (right panel). LC, loading control. Results in (AD) represent the means of three independent experiments.
Figure 8
Figure 8
TOR partial depletion or eIF3h C-terminal deletion impair reinitiation after uORF translation in mesophyll protoplasts. (A) WT or TOR RNAi protoplasts were co-transformed with reporter plasmids, which are shown in Figure 7B. Activity of GUS synthesized in protoplasts transfected with pmonoGUS was set as 100% (245 000 RFU). GUS/GFP ratios were calculated and shown as open (for pmonoGUS/pmonoGFP) and black bars (for pARF5-GUS/pmonoGFP). (B) eif3h-1 mesophyll protoplasts were co-transformed with reporters shown in Figure 7B with or without plasmid expressing eIF3h as indicated. Activity of GUS synthesized in protoplasts transfected with pmonoGUS was set as 100% (110 000 RFU). eIF3h and its phosphorylation status were assayed by immunoblotting (top panels). (C) WT mesophyll protoplasts were co-transfected with plasmids expressing eIF3h, or eIF3h-S178D, or S178A in addition to reporters shown in Figure 7B as indicated. The value of pmonoGUS expression was set as 100% (205 000 RFU). eIF3h and its phosphorylation status at S178 were assayed by immunoblotting (top panels). Results shown in (AC) represent the means obtained in three independent experiments. Source data for this figure is available on the online supplementary information page.
Figure 9
Figure 9
Torin-1 interferes with root gravitropic responses of wild type and abolishes that of TOR RNAi plants. (A) WT, TOR RNAi, and eif3h-1 seedlings grown vertically for 7 dag. (B) TOR RNAi/Torin-1 and WT/Torin-1 plants display agravitropic phenotype. Seedlings described in (A) were grown on medium without (Mock) or with 250 nM Torin-1 and analysed 24 h after gravity stimulation. The orientation of root growth of 12 seedlings was measured by assigning to 1 of 12 30° sectors; the length of each bar represents the percentage of seedlings showing this direction of root growth within the sector. (C) Seven-dag seedlings homozygous for the DR5:GFP construct were analysed 4 h after gravity (g) stimulation on control medium without (Mock) and with 250 nm Torin-1. Scale bars, 25 mm. (D) Proposed scheme of auxin-responsive TOR function in reinitiation after uORF translation (see text for details). Torin-1 application, or TOR deficiency, or eIF3h C-terminal deletion inhibit reinitiation at the steps indicated.

Comment in

  • TOR tour to auxin.
    Bögre L, Henriques R, Magyar Z. Bögre L, et al. EMBO J. 2013 Apr 17;32(8):1069-71. doi: 10.1038/emboj.2013.69. Epub 2013 Mar 22. EMBO J. 2013. PMID: 23524852 Free PMC article.

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