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Boric acid intercepts 80S ribosome migration from AUG-stop by stabilizing eRF1

Nature Chemical Biology (2024)Cite this article

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Abstract

In response to environmental changes, cells flexibly and rapidly alter gene expression through translational controls. In plants, the translation of NIP5;1, a boric acid diffusion facilitator, is downregulated in response to an excess amount of boric acid in the environment through upstream open reading frames (uORFs) that consist of only AUG and stop codons. However, the molecular details of how this minimum uORF controls translation of the downstream main ORF in a boric acid-dependent manner have remained unclear. Here, by combining ribosome profiling, translation complex profile sequencing, structural analysis with cryo-electron microscopy and biochemical assays, we show that the 80S ribosome assembled at AUG-stop migrates into the subsequent RNA segment, followed by downstream translation initiation, and that boric acid impedes this process by the stable confinement of eukaryotic release factor 1 on the 80S ribosome on AUG-stop. Our results provide molecular insight into translation regulation by a minimum and environment-responsive uORF.

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Fig. 1: Ribosomes assembled on AUG-stop migrate into downstream RNA segments under boric acid-limited conditions.
Fig. 2: Structural overview of an 80S ribosome stalled on the minimum uORF, AUG-stop, in the NIP5;1 5′ UTR in the presence of boric acid.
Fig. 3: Boric acid stabilizes eRF1 on the A site, establishing robust binding to the 80S ribosome with tRNAi in the P site.
Fig. 4: Hydrolysis of Met-tRNAi in the boric acid-stalled ribosomes.
Fig. 5: Scheme of the translation regulation process on AUG-stop in NIP5;1 in response to boric acid.

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Data availability

The ribosome profiling data (GEO GSE189222) and TCP-seq data (GEO GSE223755) were deposited in the National Center for Biotechnology Information. The cryo-EM maps and structural coordinates of the wheat ribosomes generated for this study were deposited in the Electron Microscopy Data Bank and PDB and are available under the following accession codes: high boric acid 80S complex (EMD-35634, PDB 8IP8), high boric acid 40S complex (EMD-35635, PDB 8IP9), high boric acid 40S complex with eIF2 binding (EMD-35636), high boric acid/CHX complex (EMD-35637, PDB 8IPA) and low boric acid/CHX complex (EMD-35638, PDB 8IPB). Source data are provided with this paper.

Code availability

The code for the deep-sequencing data analysis has been deposited in Zenodo (https://doi.org/10.5281/zenodo.8369134).

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Acknowledgements

This work was supported, in part, by the Ministry of Education, Culture, Sports, Science and Technology of Japan (JP19H05637 and JP18H05490 (T.F.), JP18K06278 (M.T.), JP19H02959 and JP20H05784 (S.I.) and JP19H03172 and JP21H05281 (T.I.)), the Japan Science and Technology Agency (JPMJPR20EG; T.Y.), the Japan Agency for Medical Research and Development (AMED; JP23gm1410001; S.I. and T.I.) and RIKEN (Pioneering project ‘Biology of Intracellular Environments’; M.S., S.I. and T.I.). N.S. was a JSPS Overseas Research Fellow. H. Saito was a Junior Research Associate of RIKEN. This work was also supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research from AMED; JP20am0101082, support number 0245), Support Unit for Bio-Material Analysis, RIKEN CBS Research Resources Division for Sanger sequencing and the supercomputer HOKUSAI Sailing Ship at RIKEN for computations. The cryo-EM with the CRYO ARM 300 II (powered by AMED research grant JP20am0101095) was provided by the Advanced Research Center for Innovations in Next-Generation Medicine, Tohoku University. Some of the cryo-EM experiments were performed at the RIKEN Yokohama cryo-EM facility (Yokohama).

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Author notes

  1. These authors contributed equally: Mayuki Tanaka, Takeshi Yokoyama, Hironori Saito.

Authors and Affiliations

  1. Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan

    Mayuki Tanaka, Naoyuki Sotta & Toru Fujiwara

  2. RIKEN Center for Biosystems Dynamics Research, Tsurumi-ku, Yokohama, Japan

    Takeshi Yokoyama, Madoka Nishimoto, Kengo Tsuda, Hideki Shigematsu, Mikako Shirouzu & Takuhiro Ito

  3. Graduate School of Life Sciences, Tohoku University, Sendai, Japan

    Takeshi Yokoyama

  4. Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan

    Hironori Saito & Shintaro Iwasaki

  5. RIKEN Cluster for Pioneering Research, Wako, Japan

    Hironori Saito & Shintaro Iwasaki

  6. Life Science Research Infrastructure Group, RIKEN SPring-8 Center, Sayo, Japan

    Hideki Shigematsu

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Contributions

M.T., T.Y., H. Saito, M.S., S.I., T.I. and T.F. designed the concept. M.T., T.Y. and H. Shigematsu established the methodology. M.T. performed western and northern blotting and prepared the cryo-EM samples. T.Y. collected the cryo-EM data and solved the structures. H. Saito performed ribosome profiling and TCP-seq, and M.N. conducted the purification of eRFs and the tRNA aminoacylation assay. M.T., T.Y., H. Saito, K.T., N.S., H. Shigematsu, T.I., M.S. and T.F. performed formal analyses. M.T., T.Y., N.S., S.I., T.I. and T.F. wrote the original draft. M.T., T.Y., H. Saito, M.N., K.T., N.S., H. Shigematsu, M.S., S.I., T.I. and T.F wrote, reviewed and edited the paper. M.T., T.Y., H. Saito and S.I. visualized the results. M.S., S.I., T.I. and T.F. supervised the project.

Corresponding authors

Correspondence to Shintaro Iwasaki, Takuhiro Ito or Toru Fujiwara.

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Extended data

Extended Data Fig. 1 Characterization of ribosome footprints from reporter mRNA translated in vitro.

(a) Relative luminescence for the reporter mRNA bearing the NIP5;1 5′-UTR. In addition to the wild-type NIP5;1 5′-UTR with AUG-UAA, a mutated (AUC-UAA) reporter was tested with the indicated concentrations of boric acid supplementation. Means (points) ± standard deviations (errors) from three replicates are shown. (b and e) Length distribution of ribosome footprints mapped to the main ORF (b) and the inter-ORF region (e). (c and f) Frame position of the 5′ end of footprints (27 nt) mapped to the main ORF (c) or the inter-ORF region (f). Means (bars) from two replicates are shown. (d and g) A and T contents in the main ORF sequence (d) and the inter-ORF sequence (g). (h) Sucrose density gradient centrifugation for crosslinked ribosomal complexes, used for TCP-Seq. (i) The distribution of 5′ ends of footprints in TCP-Seq. For b-c, e, f, and h-i, the data from two biologically independent replicates are shown. RPM, reads per million mapped reads.

Extended Data Fig. 2 Image processing scheme for the cryo-EM reconstruction of the 80S stalled complex on AUG-stop in +B conditions.

(a) Details of the image processing to obtain cryo-EM maps of the 80S stalled on AUG-stop in +B conditions and 40S initiation complexes with or without eIF2 binding. Of the 1,407 K particles initially 2D classified into 80S and 40S subgroups, 367 K particles sorted to 80S structures were auto-refined to obtain the consensus reconstruction. Particles were further 3D classified based on the alignment information from the consensus structure. Afterwards, 246 K particles were sorted to 80S ribosomes in complex with P-tRNA, E-tRNA, mRNA, and eRF1. The focused classification on eRF1 with residual signal subtraction was performed to obtain particles containing eRF1. The final reconstruction with 96 K particles was at a 2.9 Å resolution. In total, 181 K particles corresponding to 40S structures from the 2D classification were auto-refined to obtain the consensus reconstruction. 40S particles were 3D classified based on the alignment information from the consensus structure. Subsequently, 53 K particles were sorted to 40S ribosomes in complex with P-tRNA, mRNA, and eIF2. Focused classification on tRNAi and eIF2 with residual signal subtraction was performed to classify particles into subgroups with or without eIF2 binding. As a result, 41 K particles without eIF2 and 12 K particles with eIF2 were reconstructed at 3.0 Å and 3.7 Å, respectively. (b-c) Resolution (gold-standard FSC) curves of three reconstructions (b) and models vs. cryo-EM maps (c) are shown. (d) Color-coded local resolution distributions of the cryo-EM map of the 80S complex from this dataset.

Extended Data Fig. 3 40S complexes reconstructed from sorted subgroups of the 3D classification.

(a and b) Cryo-EM structures of the wheat 40S ribosome in complex with a tRNAi and an mRNA bearing AUG-stop (a) and that with the additional eIF2 binding (b). The contour level of the density corresponding to eIF2 in b is lowered to show entire structure of eIF2.

Extended Data Fig. 4 Cryo-EM density of tRNA in the P site represents a tRNAi-specific structural feature.

(a) Schematic diagram of tRNAi (left) and an elongator tRNAMet (right) for depicting the differences between the two tRNA species that charge methionine. (b) A close-up view of G31 and C39 base-pairing in the cryo-EM reconstruction. The density fits well to an atomic model of tRNAi, rather than the elongator tRNAMet, which has two pseudouridines in the corresponding nucleotide positions (ψ31 and ψ39).

Extended Data Fig. 5 The GGQ motif on the tip of the M domain of eRF1 in +B conditions is located near the CCA-end of tRNAi.

Isolated cryo-EM densities corresponding to the CCA-end of tRNAi (green) and the GGQ motif of eRF1 (blue) are shown in their surrounding ribosomal environments.

Extended Data Fig. 6 Image processing scheme of the cryo-EM reconstruction of the 80S stalled complex on AUG-stop in +B/ − B conditions with CHX.

(a) Details of image processing to obtain the cryo-EM map of the 80S stalled on AUG-stop in +B conditions with CHX addition. First, 131 K particles were initially 2D classified to obtain 80S particles. The 98 K particles sorted to 80S structures were auto-refined to obtain the consensus reconstruction. Particles were further 3D classified based on the alignment information from the consensus structure. 80S ribosomes in complex with P-tRNA, mRNA, eRF1, and CHX were selected. Focused classification on eRF1 with residual signal subtraction was performed to obtain particles containing eRF1. The final reconstruction with 69 K particles was at a 3.4 Å resolution. (b) Details of image processing to obtain the cryo-EM map of the 80S stalled on AUG-stop in −B conditions with CHX addition. The picked 220 K particles were initially 2D classified to obtain 80S particles, and then 92 K particles sorted to 80S structures were auto-refined to obtain the consensus reconstruction. Particles were further 3D classified based on the alignment information from the consensus structure. 80S ribosomes in complex with P-tRNA, mRNA, and CHX were selected. The focused classification on the fragmented eRF1 density with residual signal subtraction was performed. The obtained 52 K particles containing some density on the A site were reconstructed at a 3.4 Å resolution. (c-d) Resolution (gold-standard FSC) curves of two reconstructions (c) and models vs. cryo-EM maps (d) are shown. (e-f) Color-coded local resolution distributions of the cryo-EM map of the 80S complex from these datasets.

Extended Data Fig. 7 Cycloheximide binding to the E site sterically clashes with A76 of a deacylated tRNA bound to the E site.

(a-b) Cryo-EM densities of cycloheximide (a) and the CCA-end (b) and their corresponding atomic models are shown in the 60S environment. (c) Superimposition of the two models, showing the steric clash of cycloheximide with tRNA.

Extended Data Fig. 8 Purification of recombinant Arabidopsis eRF1-1 and eRF3 proteins.

CBB staining of the indicated recombinant proteins. The expected molecular weights of eRF1-1 and eRF3 are 48.7 kDa and 60.5 kDa, respectively. The lane of purified eRF3 was on the same gel but the unrelated lanes in the middle were omitted without changing the Y-axis position of the bands. The similar staining image was obtained repeatedly (3 replicates).

Extended Data Fig. 9 Characterization of Met-tRNAi hydrolysis on ribosomes stalled on AUG-stop.

(a and b) The same as Fig. 4d but with titrated amounts of eRF1/eRF3 (a) and along the time course (b). For a, 0, 1, 10, and 100 ng of eRF1/eRF3 were used and incubated for 30 min in the presence of boric acid. For b, reactions were incubated for 0, 5, 10, 30 and 60 min with 300 ng eRF1/eRF3 in the presence of boric acid. For a, means (bars) of two biological replicates (points) are shown. For b, means (large points) of two biologically independent replicates (small points) are shown.

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Supplementary Fig. 1 and Tables 1 and 2.

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Source Data Fig. 3

Uncropped western blots in Fig. 3d,e.

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Uncropped northern blots in Fig. 4a,c,d.

Source Data Figs. 3 and 4

Statistical source data for Figs. 3 and 4.

Source Data Extended Data Fig. 9

Uncropped northern blot in Extended Data Fig. 9a,b.

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Tanaka, M., Yokoyama, T., Saito, H. et al. Boric acid intercepts 80S ribosome migration from AUG-stop by stabilizing eRF1. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-023-01513-0

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