Translation is a fundamental step in gene expression that regulates multiple developmental and stress responses. One key step of translation initiation is the association between eIF4E and eIF4G. This process is regulated in different eukaryotes by proteins that bind to eIF4E; however, evidence of eIF4E-interacting proteins able to regulate translation is missing in plants. Here, we report the discovery of CERES, a plant eIF4E-interacting protein. CERES contains an LRR domain and a canonical eIF4E-binding site. Although the CERES–eIF4E complex does not include eIF4G, CERES forms part of cap-binding complexes, interacts with eIF4A, PABP and eIF3, and co-sediments with translation initiation complexes in vivo. Moreover, CERES promotes translation in vitro and general translation in vivo, while it modulates the translation of specific mRNAs related to light and carbohydrate response. These data suggest that CERES is a non-canonical translation initiation factor that modulates translation in plants.
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The data that support the findings of this study are available from the corresponding author upon reasonable request. In addition, super-resolution profile data have been deposited in the Gene Expression Omnibus database (GEO-NCBI) (http://www.ncbi.nlm.nih.gov/geo/) with the accession code GSE124290. Source data for Figs. 2–4 and Extended Data Fig. 5 are presented with the paper.
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This research has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement no. 260468 to M.M.C. and from the grant S2013-ABI2748 from CAM. In addition, this work has been partially financial supported by RTI2018-095946-B100 from MICIU and by ‘Severo Ochoa Programme for Centres of Excellence in R&D’ from the Agencia Estatal de Investigación of Spain (grant SEV-2016-0672 (2017-2021) to the CBGP). In the frame of this last programme, R.T. was supported with a postdoctoral contract. We are indebted to J. Berlanga for the use of the gradient fractionation system and to P. Olivares and I. Díaz for assistance. We deeply thank G. Hernández, A. Ferrando, J. Berlanga and F. García-Arenal for helpful comments on the manuscript.
The authors declare no competing interests.
Peer review information Nature Plants thanks Gallois Jean-Luc and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
(a) Yeast two-hybrid assays to analyse CERES interaction with AteIF(iso)4E. The proteins fused to the Gal4-BD and Gal4-AD that were co-expressed in the AH109 strain are shown on the left of the panel. Independent co-transformants were tested for growth in non-selective medium (-Leu-Trp) or prototrophy-selective medium (-Leu, -Trp, -His) in the presence of 3-AT or in the absence of Ade. The constructs expressing the bare Gal4-BD and Gal4-AD were used as controls (-). (b) CERES interacts with AteIF4E1 and At(iso)4E in vivo. Protein extracts (crude extracts) from N. benthamiana leaves transiently expressing, under the control of the 35S promoter, different combinations of Flag–CERES, HA–AteIF4E1 and HA–AteIF(iso)4E were subjected to immunoprecipitation using anti-Flag beads. The presence of the different proteins in the crude extracts and in the eluted fractions from CERES immunoprecipitations (IP:α-Flag) was analysed by western-blot using anti-HA and anti-Flag antibodies. The experiments in (a-b) were repeated independently three times with similar results.
Extended Data Fig. 2 Western-blot to analyse the size of the fusion proteins and the accumulation of the proteins of interest in Fig. 1c and Fig. 2d.
(a) Western-blot of extracts from N. benthamiana leaves expressing the constructs pCERES::CERES–GFP or p35S::GFP (in this case two extracts with different expression level of GFP were included) (left panel) or the constructs p35S::RedFP and p35S::RedFP–AteIF4E1 (right panel) using the anti-GFP and anti-RFP, respectively. The Coomassie staining is provided as loading control of the assay. (b) Western-blot of yeast extracts that expressed from the pDEST-GADT7 and pDEST-GBKT7 vectors the different proteins of interest. These vectors allow the fusion of the proteins to the Gal4-AD and the HA and to the Gal4-BD and the c-Myc epitopes, respectively. The fusion proteins were detected using the anti-HA and anti-Myc antibodies. Possible degradation products are marked by an asterisk. The experiments in (a-b) were repeated independently twice with similar results.
The relative expression of CERES mRNA was analysed in 7-day-old whole seedlings, 10-day-old roots and 4-week-old leaves, stems and open flowers. Fold change values, shown as means ± SD (n = 4 independent experiments), are related to the expression in seedlings that was arbitrarily assigned value 1 after normalisation with the calibrator gene UBC.
Extended Data Fig. 4 CERES forms part of cap-binding complexes in vitro in the presence of AteIF4E1 and AteIF(iso)4E.
Recombinant AteIF4E1, AteIF(iso)4E and CERES fused with GST were expressed in E.coli (crude extract). These extracts were combined as detailed in the figure (using in all cases a higher amount (8-fold) of recombinant CERES) and subjected to a 7-methyl-GTP chromatography. The corresponding eluates were analysed by western-blot using a commercial anti-GST antibody. This experiment was repeated independently twice with similar results.
(a-b) Schematic genomic organisation of CERES. Exons are indicated as rectangles. The triangles mark the position of the T-DNA insertions in the ceres-1 (a) and ceres-2 (b) mutants. (b) Schematic organisation of CERES´ CDS in ceres-2. This mutant shows an aberrant splicing event that introduces a premature stop codon (PTC) in its sequence. (c) Analysis of CERES expression in 15-day-old seedlings from Col-0, ceres-1 and ceres-2 by RT-qPCR. Expression values are shown as mean ± SEM from n = 6 independent samples. These values are related to the value of Col-0 that was arbitrarily assigned value 1 after normalization with the calibrator gene ACT-2. (d) Western-blot analysis of CERES accumulation in Col-0 and in ceres mutants using specific anti- CERES antibodies generated in the laboratory. The Ponceau staining of the membrane is provided as loading control. This experiment was repeated independently four times with similar results.
(a) Correlation plots of the RFP (R1, R2 and R3) and total RNA (T1, T2, T3) samples of each genotype used for the super-resolution ribosome profiling analysis. (b) Boxplots of RFP coverage on 5’ UTR, CDS and 3’ UTR regions. RFP reads from the replicates (n = 3 independent experiments) were grouped for each genotype. The middle bars represent the median, while the bottom and top of each box represent the 25th and 75th percentiles, respectively,and the whiskers extend to 1.5 times the interquartile range. Dots are outliers. Median value for reads on 3’UTRs is 0.
Extended Data Fig. 7 Periodicity analysis (cumulative plots) of RFP reads ranging from 24 to 30 nt in length.
The first nucleotide of each footprint is used to represent its location on the transcript. The three reading frames are shown in red, blue and green. As representative data, the analysis of RFP from Col-0 replicate 1 is shown. (b) Percentage of RFP reads derived from the 24-30 nt fragments used in the study.
Extended Data Fig. 8 Coverage of RFP and total RNA reads on selected genes in Col-0 and ceres mutants.
The scale of the reads for each gene is indicated in the upper left. Based on the total RNA reads, the most prevalent predicted genomic organisation is shown in the bottom panel of each gene. Exons are indicated as dark blue rectangles and introns as dark blue lines. In this analysis the reads of each genotype from n = 3 independent experiments were combined.
Extended Data Fig. 9 ceres mutants do not seem to show an altered phenotype in response to mannitol.
(a) Representative growth of Col-0, ceres-1 and ceres-2 seedlings in medium lacking mannitol (control) or supplemented with 300 mM mannitol for 7 days. (b) Close-up views of the upper panel. (c) Percentage of seedlings from Col-0, ceres-1 and ceres-2 that develop green and expanded cotyledon in the presence of mannitol. n = 3 independent experiments were analysed. Values are shown as means ± SEM. No statistical difference between Col-0 and ceres mutants (p < 0.05) using one-way ANOVA analysis was observed. The scale bars in (a) and (b) correspond to 1.5 cm and 7.5 mm, respectively.
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Toribio, R., Muñoz, A., Castro-Sanz, A.B. et al. A novel eIF4E-interacting protein that forms non-canonical translation initiation complexes. Nat. Plants 5, 1283–1296 (2019). https://doi.org/10.1038/s41477-019-0553-2