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The eRF1 degrader SRI-41315 acts as a molecular glue at the ribosomal decoding center

Nature Chemical Biology (2024)Cite this article

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Abstract

Translation termination is an essential cellular process, which is also of therapeutic interest for diseases that manifest from premature stop codons. In eukaryotes, translation termination requires eRF1, which recognizes stop codons, catalyzes the release of nascent proteins from ribosomes and facilitates ribosome recycling. The small molecule SRI-41315 triggers eRF1 degradation and enhances translational readthrough of premature stop codons. However, the mechanism of action of SRI-41315 on eRF1 and translation is not known. Here we report cryo-EM structures showing that SRI-41315 acts as a metal-dependent molecular glue between the N domain of eRF1 responsible for stop codon recognition and the ribosomal subunit interface near the decoding center. Retention of eRF1 on ribosomes by SRI-41315 leads to ribosome collisions, eRF1 ubiquitylation and a higher frequency of translation termination at near-cognate stop codons. Our findings reveal a new mechanism of release factor inhibition and additional implications for pharmacologically targeting eRF1.

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Fig. 1: SRI-41315 inhibits protein synthesis and induces eRF1 ubiquitylation in vitro.
Fig. 2: Cryo-EM structure of SRI-41315 on a terminating ribosome.
Fig. 3: Impact of SRI-41315 on eRF1 and the decoding center.
Fig. 4: SRI-41315 induces translation termination at cryptic stop codons.

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Data availability

EM maps and models are available under accession numbers PDB 8SCB, EMD-40344 and EMD-40345. All other data supporting the findings of this study are within the article and its Extended Data. Raw data files needed in other formats are available from the corresponding author upon reasonable request. Additional data used in this study are PDB 6SGC, 3JAG, 6MTE and 6XA1. Source data are provided with this paper.

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Acknowledgements

Cryo-EM screening and data collection were performed at the Harvard Center for Cryo-Electron Microscopy (HC2EM). Data processing was supported by SBGrid. We thank A. Brown, Y. Peng and Shao laboratory members for helpful discussions. This work was supported by the Packard Foundation (S.S.), NIH DP2GM137415 (S.S.) and the UCSF Invent Fund (J.T.). M.C.J.Y. was supported by the American Heart Association (predoctoral fellowship 287375208). K.O. was supported by a UCSF Genentech Fellowship.

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Author notes

  1. These authors contributed equally: João P. L. Coelho, Matthew C. J. Yip.

Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    João P. L. Coelho, Matthew C. J. Yip & Sichen Shao

  2. Chemistry and Chemical Biology Graduate Program, University of California San Francisco, San Francisco, CA, USA

    Keely Oltion

  3. Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA

    Jack Taunton

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  1. João P. L. Coelho
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Contributions

J.P.L.C., M.C.J.Y. and S.S. performed biochemical analyses. K.O. performed flow cytometry experiments. M.C.J.Y. collected and processed cryo-EM data. S.S. and J.T. conceived the project. S.S. wrote the paper with input from all authors.

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Correspondence to Sichen Shao.

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J.T. is a founder of Kezar Life Sciences and Terremoto Biosciences and is a scientific advisor to Entos. The other authors declare no competing interests.

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Nature Chemical Biology thanks Yury Polikanov, Daniel Wilson and the 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 SRI-41315 in an in vitro translation system.

a, Effect of SRI-41315 on protein synthesis. Representative titrations of SRI-41315 into in vitro translation reactions of a radiolabeled 3xFlag-tagged model protein ending with the indicated stop codon or a nonstop (NS) control, analyzed by SDS-PAGE and autoradiography on film (top) or by phosphorimaging (bottom). NC, expected nascent protein chain; NC-tRNA, nonhydrolyzed peptidyl-tRNA adduct. Orange dots, smaller products specifically observed with SRI-41315, representative of 5 replicates with similar results. b, SRI-41315 does not trap nascent proteins on ribosomes. In vitro translation reactions as in panel a without or with 0.5 µM eRF1(AAQ) and/or 100 µM SRI-41315 added after 5 min were size fractionated on 10-50% sucrose gradients. The total (T) and eleven fractions collected from the top of each gradient were analyzed by SDS-PAGE and autoradiography. Note: eRF1(AAQ) but not SRI-41315 retains NC-tRNAs and NCs hydrolyzed from tRNAs during SDS-PAGE in ribosomal fractions, representative of 3 replicates with similar results.

Source data

Extended Data Fig. 2 SRI-41315 traps ubiquitylated eRF1 on ribosomes.

a, RNF14 levels in different mammalian lysates. Two-fold dilutions of rabbit reticulocyte lysate (RRL) or HEK293T cell lysate analyzed by SDS-PAGE and immunoblotting. Note: low levels of RNF14 in RRL relative to GCN1 and ribosomal proteins, representative of 2 replicates with similar results. b, RNF14 mediates eRF1 ubiquitylation with SRI-41315. In vitro translation reactions containing 10 µM His-tagged ubiquitin without or with 50 nM wildtype (WT) or catalytically inactive (C417A) recombinant RNF14 (rRNF14) and 100 µM SRI-41315 were analyzed directly (total) or after denaturing pulldowns of His-tagged ubiquitin (His-Ub PD) by SDS-PAGE and immunoblotting. Note: WT but not C417A rRNF14 enhances SRI-41315-dependent ubiquitylation of eRF1, representative of 3 replicates with similar results. Residual eRF1 ubiquitylation is likely due to endogenous RNF14 in RRL. c, Titration of SRI-41315 into in vitro translation reactions containing 50 nM recombinant RNF14 analyzed directly (total) or after His-Ub PD by SDS-PAGE and immunoblotting, representative of 3 replicates with similar results. d, eRF1 ubiquitylation is slower than translation. Timecourses assaying eRF1 ubiquitylation in the presence of 50 nM recombinant RNF14, 10 µM His-tagged ubiquitin, and 25 µM SRI-41315 (top) compared to timecourses of radiolabeled nascent protein (NC) synthesis (bottom) in in vitro translation reactions, representative of 2 replicates with similar results. e, SRI-41315 traps eRF1 on ribosomes. Translation reactions as in Fig. 1c were size fractionated over 10-50% sucrose gradients. A total (T) sample and eleven fractions collected from the top of each gradient were analyzed directly or after His-Ub PD by SDS-PAGE and immunoblotting for eRF1. Note: SRI-41315 retains both unmodified and ubiquitylated eRF1 in ribosomal fractions, representative of 3 replicates with similar results.

Source data

Extended Data Fig. 3 SRI-41315 recruits collision sensors to ribosomes.

a,b, Effects of SRI-41315 in cells. Lysates of Flp-In 293 T-REx cells treated without or with 30 µM SRI-41315, 1.8 µM emetine, a concentration of the translation elongation inhibitor demonstrated to cause ribosome collisions, and/or 1 µM MLN-7243, an inhibitor of the E1 ubiquitin-activating enzyme, for 2 hr were analyzed by SDS-PAGE and immunoblotting a, directly or b, after size fractionation on sucrose gradients, representative of 2 replicates with similar results. Note: ubiquitylated eRF1 (Ub-eRF1) is detected specifically with SRI-41315 and suppressed by MLN-7243. SRI-41315 and the low dose of emetine both lead to the recruitment of the ribosome collision sensor EDF1 to ribosomal fractions. c, SRI-41315 induces the recruitment of EDF1 to ribosomes in vitro. SDS-PAGE and immunoblotting for the ribosome collision sensor EDF1 in total, soluble, or ribosomal fractions from translation reactions containing the indicated concentrations of SRI-41315, representative of 2 replicates with similar results. d, SRI-41315 induces eRF1 degradation in Flp-In 293 T-REx cells after 20 hr, representative of 3 replicates with similar results.

Source data

Extended Data Fig. 4 Cryo-EM data processing.

a, Ribosome-nascent protein complexes (RNCs) from translation reactions containing 0.5 µM eRF1(AAQ) and 100 µM SRI-41315 added at 5 min were affinity purified via the 3xFlag tag encoded in the nascent chain (NC) and analyzed by SDS-PAGE and Coomassie staining (top) or immunoblotting (bottom). NC-tRNA, nonhydrolyzed peptidyl-tRNA adduct. b, Representative cryo-EM micrograph of RNCs from panel a. c, Summary of cryo-EM data processing and classification strategy.

Source data

Extended Data Fig. 5 Quality of cryo-EM maps and model.

a, Fourier shell correlation (FSC) vs. resolution (1/Å) curves for the indicated cryo-EM maps. b, The indicated cryo-EM map colored by local resolution. c, Model vs. map FSC curves. d, Density of the defined nascent protein sequence in the ribosomal exit tunnel in the sharpened cryo-EM map contoured at 2.8σ. e, The cryo-EM map of the SRI-41315 binding site as in Fig. 2b colored by local resolution (left) or by entity (right). Magnesium ions are in green. f, Coordination of the magnesium ion at the SRI-41315 binding site compared to density in the sharpened cryo-EM map contoured at 4.0σ.

Extended Data Fig. 6 eRF1 secondary structure and alignments.

Sequence alignments of eRF1 from the indicated species (H. sapiens UniProt P62495; O. cuniculus UniProt P62497; M. musculus UniProt Q8BWY3; X. laevis UniProt P35615; D. rerio UniProt Q803E5; D. melanogaster UniProt Q9VPH7; C. elegans UniProt O16520; S. cerevisiae UniProt P12385) with secondary structure designations above colored based on their presence in the N (blue), M (green), and C (orange) domains of eRF1. In the accommodated state of eRF1, α8 of the M domain and α9 of the C domain form a continuous helix (yellow). eRF1 also contains a minidomain insertion (gray) in the C domain that is not present in structurally similar decoding factors. In the N domain, Met51 (purple arrowhead) involved in SRI-41315 binding, as well as the NIKS and YxCxxxF motifs (gray lines) and Glu55 (gray arrowhead) involved in stop codon recognition, are indicated below. Note: human, rabbit, and mouse eRF1 are 100% identical. Part of the C-terminal extension in C. elegans eRF1 is not shown.

Extended Data Fig. 7 Structural comparisons.

a, Validation of SRI-41315 density. The model of the SRI-41315 binding site as in Fig. 2b docked into cryo-EM maps of the rabbit ribosome bound to eRF1(AAQ) without SRI-41315 (EMD-3038; left), of the human ribosome bound to eRF1 trapped by PF846 (EMD-22085; middle), or generated from particles selected for occupancy of the recycling factor ABCE1 (right), contoured at the indicated levels. Note: the left and middle maps lack SRI-41315, while the right map retains strong density corresponding to SRI-41315 coexisting with ABCE1 binding. b, The overall conformation of accommodated eRF1 is unchanged with SRI-41315. The model of eRF1(AAQ) bound to SRI-41315 (purple) aligned to eRF1(AAQ) without SRI-41315 (pink; PDB 3JAG) or eRF1 trapped on ribosomes by PF846 (blue; 6XA1). The N, M, and C domains (left), the GGQ motif, and the SRI-41315 binding site (right) are indicated. c, Docking of related small molecule eRF1 degraders in the SRI-41315 binding site. d, SRI-41315 binding region of eRF1 (PDB 1DT9) colored by conservation. Note: Met51 is less conserved than Tyr125 and other residues required for stop codon decoding. e, SRI-41315 does not change ABCE1 conformation. Alignment of ABCE1 on termination complexes without (pink; PDB 3JAG) or with (dark blue) SRI-41315. Iron-sulfur clusters are colored by heteroatoms. f, ABCE1 is slightly stabilized on ribosomes with SRI-41315. Total, soluble, and ribosomal fractions of translation reactions without or with 100 µM SRI-41315 as in Fig. 1c were analyzed by SDS-PAGE and immunoblotting for ABCE1, representative of 3 replicates with similar results.

Source data

Extended Data Fig. 8 Flow cytometry analysis.

a, Cells were initially gated by FSC-A vs. SSC-A to exclude debris. b, Cells from the gate in panel a were gated by FSC-A vs. FSC-W to exclude doublets. c, Cells from the gate in panel b were gated by GFP positivity (left) as judged by comparison to an untransduced reference (right) to exclude untransduced cells from analysis.

Extended Data Fig. 9 Cryptic stop codon analysis.

a, Transcript (top line) and polypeptide (bottom line) sequence of the reporter designed to test translation termination at cryptic stop codons. The reporter contains an N-terminal 8xHis tag (blue), a modified calmodulin sequence (light orange), the test codon position (dark orange; stop1), a modified sequence encoding the autonomously folding villin headpiece (VHP) domain followed by the cytosolic domain of Sec61β (light green), and the UAA stop codon (black; stop2). The reporter sequence was modified to remove all codons, except for one UGG codon (pink), that start with a U and contain a purine (A/G) in the second and/or third positions. Additional cryptic codons that start with a C and contain purines in the second and third positions (purple) were tested by mutations (see panel c). b, SRI-41315 induces translation termination at specific cryptic stop codons. Quantification of the ratio of stop1 to stop2 products from reporters containing UUA (green) or UAU (orange) in the test codon position synthesized in vitro with increasing concentrations of SRI-41315. The normalized average (line) and individual ratios (dots) from three independent experiments are shown. c, Mutagenesis of additional cryptic stop codons. Assays as in Fig. 4a with UGA (lane 1) or UGG (lanes 2–5) in the test codon position and additional mutation of CAG and CAA codons (Qmut; purple) upstream of the stop1 position and/or of the UGG codon (Wmut; pink) downstream of the stop1 position as indicated in panel a. Radiolabeled reporter products generated without (left) or with (right) 100 µM SRI-41315 are shown. Note: changes in products upon mutation of these codons occur specifically with SRI-41315, representative of 3 replicates with similar results. Purple dots denote products abolished by mutating the CAG/CAA codons; pink dot denotes the lower band of a doublet abolished by mutating the UGG codon. Yellow dot denotes a band with increased intensity after mutation of the CAG/CAA codons.

Source data

Supplementary information

Source data

Source Data Fig. 1

Unprocessed images of stained gels, western blots, autoradiography and phosphorimaging.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Unprocessed autoradiography image.

Source Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed images of autoradiography and phosphorimaging.

Source Data Extended Data Fig. 2

Unprocessed images of stained gels, western blots, autoradiography and phosphorimaging.

Source Data Extended Data Fig. 3

Unprocessed images of Ponceau stained blots and western blots.

Source Data Extended Data Fig. 4

Unprocessed images of stained gels and western blots.

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Unprocessed image of western blot.

Source Data Extended Data Fig. 9

Unprocessed images of autoradiography.

Source Data Extended Data Fig. 9

Statistical source data.

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Coelho, J.P.L., Yip, M.C.J., Oltion, K. et al. The eRF1 degrader SRI-41315 acts as a molecular glue at the ribosomal decoding center. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-023-01521-0

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