Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structural mechanism of angiogenin activation by the ribosome

Abstract

Angiogenin, an RNase-A-family protein, promotes angiogenesis and has been implicated in cancer, neurodegenerative diseases and epigenetic inheritance1,2,3,4,5,6,7,8,9,10. After activation during cellular stress, angiogenin cleaves tRNAs at the anticodon loop, resulting in translation repression11,12,13,14,15. However, the catalytic activity of isolated angiogenin is very low, and the mechanisms of the enzyme activation and tRNA specificity have remained a puzzle3,16,17,18,19,20,21,22,23. Here we identify these mechanisms using biochemical assays and cryogenic electron microscopy (cryo-EM). Our study reveals that the cytosolic ribosome is the activator of angiogenin. A cryo-EM structure features angiogenin bound in the A site of the 80S ribosome. The C-terminal tail of angiogenin is rearranged by interactions with the ribosome to activate the RNase catalytic centre, making the enzyme several orders of magnitude more efficient in tRNA cleavage. Additional 80S–angiogenin structures capture how tRNA substrate is directed by the ribosome into angiogenin’s active site, demonstrating that the ribosome acts as the specificity factor. Our findings therefore suggest that angiogenin is activated by ribosomes with a vacant A site, the abundance of which increases during cellular stress24,25,26,27. These results may facilitate the development of therapeutics to treat cancer and neurodegenerative diseases.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Angiogenin inhibits translation by binding to 80S ribosomes.
Fig. 2: Angiogenin’s RNase is activated after ribosome binding.
Fig. 3: Cryo-EM structures of 80S–angiogenin complexes formed with eEF1A–GDPCP and tRNA.
Fig. 4: Schematic of angiogenin activation and specificity toward tRNA induced by the 80S ribosome.

Similar content being viewed by others

Data availability

The structural models generated in this study have been deposited in the RCSB Protein Data Bank under the following accession codes: 9BDL (rabbit 80S ribosome with angiogenin), 9BDN (rabbit ribosome with angiogenin and tRNAAla) and 9BDP (rabbit ribosome with angiogenin and Ala-tRNAAla bound to eEF1A). The cryo-EM maps used to generate models in this study have been deposited in the Electron Microscopy Data Bank under the following accession codes: EMD-44461 (rabbit 80S ribosome with angiogenin), EMD-44463 (rabbit 80S ribosome with angiogenin and tRNAAla), EMD-44464 (rabbit 80S ribosome with angiogenin and Ala-tRNAAla bound to eEF1A). Additional cryo-EM maps have been deposited in the Electron Microscopy Data Bank under the following accession codes: EMD-44457 (80S ribosome with angiogenin in RRL), EMD-44458 (rabbit 80S ribosome with angiogenin, in vitro complex assembled without substrate tRNAAla), EMD-44459 (rabbit 80S ribosome with angiogenin(H13A)), EMD-44460 (rabbit 80S ribosome with angiogenin(H13A) and tRNAAla). The coordinate files used in this study are available at the PDB: 7TOR, 5EOP, 1EHZ, 5LZS, 5UYM, 1A4Y and 5RSA; or from the AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/entry/P02994; https://alphafold.ebi.ac.uk/entry/P02994)85,86. The electron density map used in this study is available from the Electron Microscopy Database (EMD-4729).

References

  1. Sheng, J. & Xu, Z. Three decades of research on angiogenin: a review and perspective. Acta Biochim. Biophy. Sin. 48, 399–410 (2016).

    Article  CAS  Google Scholar 

  2. Fett, J. W. et al. Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells. Biochemistry 24, 5480–5486 (1985).

    Article  CAS  PubMed  Google Scholar 

  3. Shapiro, R., Riordan, J. F. & Vallee, B. L. Characteristic ribonucleolytic activity of human angiogenin. Biochemistry 25, 3527–3532 (1986).

    Article  CAS  PubMed  Google Scholar 

  4. Zhang, Y. et al. Angiogenin mediates paternal inflammation-induced metabolic disorders in offspring through sperm tsRNAs. Nat. Commun. 12, 6673 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hartmann, A. et al. Hypoxia-induced up-regulation of angiogenin in human malignant melanoma. Cancer Res. 59, 1578–1583 (1999).

    CAS  PubMed  Google Scholar 

  6. Yoshioka, N., Wang, L., Kishimoto, K., Tsuji, T. & Hu, G.-F. A therapeutic target for prostate cancer based on angiogenin-stimulated angiogenesis and cancer cell proliferation. Proc. Natl Acad. Sci. USA 103, 14519–14524 (2006).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kishimoto, K. et al. Hypoxia-induced up-regulation of angiogenin, besides VEGF, is related to progression of oral cancer. Oral Oncol. 48, 1120–1127 (2012).

    Article  CAS  PubMed  Google Scholar 

  8. Greenway, M. et al. ANG mutations segregate with familial and ‘sporadic’ amyotrophic lateral sclerosis. Nat. Genet. 38, 411–413 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Aparicio-Erriu, I. & Prehn, J. Molecular mechanisms in amyotrophic lateral sclerosis: the role of angiogenin, a secreted RNase. Front. Neurosci. 6, 167 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Prehn, J. H. M. & Jirström, E. Angiogenin and tRNA fragments in Parkinson’s disease and neurodegeneration. Acta Pharmacol. Sin. 41, 442–446 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yamasaki, S., Ivanov, P., Hu, G.-F. & Anderson, P. Angiogenin cleaves tRNA and promotes stress-induced translational repression. J. Cell Biol. 185, 35–42 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ivanov, P., Emara, M. M., Villen, J., Gygi, S. P. & Anderson, P. Angiogenin-induced tRNA fragments inhibit translation initiation. Mol. Cell 43, 613–623 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Fu, H. et al. Stress induces tRNA cleavage by angiogenin in mammalian cells. FEBS Lett. 583, 437–442 (2009).

    Article  CAS  PubMed  Google Scholar 

  14. Saxena, S. K., Rybak, S. M., Davey, R. T., Youle, R. J. & Ackerman, E. J. Angiogenin is a cytotoxic, tRNA-specific ribonuclease in the RNase A superfamily. J. Biol. Chem. 267, 21982–21986 (1992).

    Article  CAS  PubMed  Google Scholar 

  15. Emara, M. M. et al. Angiogenin-induced tRNA-derived stress-induced RNAs promote stress-induced stress granule assembly. J. Biol. Chem. 285, 10959–10968 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Acharya, K. R., Shapiro, R., Allen, S. C., Riordan, J. F. & Vallee, B. L. Crystal structure of human angiogenin reveals the structural basis for its functional divergence from ribonuclease. Proc. Natl Acad. Sci. USA 91, 2915–2919 (1994).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. Harper, J. W. & Vallee, B. L. Mutagenesis of aspartic acid-116 enhances the ribonucleolytic activity and angiogenic potency of angiogenin. Proc. Natl Acad. Sci. USA 85, 7139–7143 (1988).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. Russo, N., Nobile, V., Di Donato, A., Riordan, J. F. & Vallee, B. L. The C-terminal region of human angiogenin has a dual role in enzymatic activity. Proc. Natl Acad. Sci. USA 93, 3243–3247 (1996).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. Russo, N., Shapiro, R., Acharya, K. R., Riordan, J. F. & Vallee, B. L. Role of glutamine-117 in the ribonucleolytic activity of human angiogenin. Proc. Natl Acad. Sci. USA 91, 2920–2924 (1994).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. Akiyama, Y., Tomioka, Y., Abe, T., Anderson, P. & Ivanov, P. In lysate RNA digestion provides insights into the angiogenin’s specificity towards transfer RNAs. RNA Biol. 18, 2546–2555 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Thomas, S. P., Hoang, T. T., Ressler, V. T. & Raines, R. T. Human angiogenin is a potent cytotoxin in the absence of ribonuclease inhibitor. RNA 24, 1018–1027 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Jardine, A. M. et al. Cleavage of 3′,5′-pyrophosphate-linked dinucleotides by ribonuclease A and angiogenin. Biochemistry 40, 10262–10272 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Leonidas, D. D. et al. Refined crystal structures of native human angiogenin and two active site variants: implications for the unique functional properties of an enzyme involved in neovascularisation during tumour growth. J. Mol. Biol. 285, 1209–1233 (1999).

    Article  CAS  PubMed  Google Scholar 

  24. Wu, C. C.-C., Zinshteyn, B., Wehner, K. A. & Green, R. High-resolution ribosome profiling defines discrete ribosome elongation states and translational regulation during cellular stress. Mol. Cell 73, 959–970 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Buschauer, R. et al. The Ccr4-not complex monitors the translating ribosome for codon optimality. Science 368, eaay6912 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ikeuchi, K. et al. Collided ribosomes form a unique structural interface to induce Hel2‐driven quality control pathways. EMBO J. 38, e100276 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Yan, L. L. & Zaher, H. S. Ribosome quality control antagonizes the activation of the integrated stress response on colliding ribosomes. Mol. Cell 81, 614–628 (2021).

    Article  CAS  PubMed  Google Scholar 

  28. Wlodawer, A., Borkakoti, N., Moss, D. S. & Howlin, B. Comparison of two independently refined models of ribonuclease-A. Acta Crystallogr. B 42, 379–387 (1986).

    Article  ADS  Google Scholar 

  29. Leonidas, D. D., Shapiro, R., Subbarao, G. V., Russo, A. & Acharya, K. R. Crystallographic studies on the role of the C-terminal segment of human angiogenin in defining enzymatic potency. Biochemistry 41, 2552–2562 (2002).

    Article  CAS  PubMed  Google Scholar 

  30. Shapiro, R. Structural features that determine the enzymatic potency and specificity of human angiogenin: threonine-80 and residues 58−70 and 116−123. Biochemistry 37, 6847–6856 (1998).

    Article  CAS  PubMed  Google Scholar 

  31. Su, Z., Kuscu, C., Malik, A., Shibata, E. & Dutta, A. Angiogenin generates specific stress-induced tRNA halves and is not involved in tRF-3–mediated gene silencing. J. Biol. Chem. 294, 16930–16941 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. St Clair, D. K., Rybak, S. M., Riordan, J. F. & Vallee, B. L. Angiogenin abolishes cell-free protein synthesis by specific ribonucleolytic inactivation of ribosomes. Proc. Natl Acad. Sci. USA 84, 8330–8334 (1987).

    Article  ADS  Google Scholar 

  33. Akiyama, Y. et al. Selective cleavage at CCA ends and anticodon loops of tRNAs by stress-induced RNases. Front. Mol. Biosci. 9, 791094 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  34. Cristodero, M. & Polacek, N. The multifaceted regulatory potential of tRNA-derived fragments. Non-coding RNA Invest. https://doi.org/10.21037/ncri.2017.08.07 (2017).

  35. Blanco, S. et al. Stem cell function and stress response are controlled by protein synthesis. Nature 534, 335–340 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. Skorupa, A. et al. Motoneurons secrete angiogenin to induce RNA cleavage in astroglia. J. Neurosci. 32, 5024–5038 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Pizzo, E. et al. Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival. J. Cell Sci. 126, 4308–4319 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Saikia, M. et al. Genome-wide identification and quantitative analysis of cleaved tRNA fragments induced by cellular stress. J. Biol. Chem. 287, 42708–42725 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Papageorgiou, A. C., Shapiro, R. & Acharya, K. R. Molecular recognition of human angiogenin by placental ribonuclease inhibitor-an X-ray crystallographic study at 2.0 A resolution. EMBO J. 16, 5162–5177 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Berthelot, F., Bogdanovsky, D., Schapira, G. & Gros, F. Interchangeability of factors and tRNA’s in bacterial and eukaryotic translation initiation systems. Mol. Cell. Biochem. 1, 63–72 (1973).

    Article  CAS  PubMed  Google Scholar 

  41. Chen, J., Sawyer, N. & Regan, L. Protein–protein interactions: general trends in the relationship between binding affinity and interfacial buried surface area. Protein Sci. 22, 510–515 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Hallahan, T. W., Shapiro, R. & Vallee, B. L. Dual site model for the organogenic activity of angiogenin. Proc. Natl Acad. Sci. USA 88, 2222–2226 (1991).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  43. Moroianu, J. & Riordan, J. F. Identification of the nucleolar targeting signal of human angiogenin. Biochem. Biophys. Res. Commun. 203, 1765–1772 (1994).

    Article  CAS  PubMed  Google Scholar 

  44. Bradshaw, W. J. et al. Structural insights into human angiogenin variants implicated in Parkinson’s disease and amyotrophic lateral sclerosis. Sci. Rep. 7, 41996 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  45. Thiyagarajan, N., Ferguson, R., Subramanian, V. & Acharya, K. R. Structural and molecular insights into the mechanism of action of human angiogenin-ALS variants in neurons. Nat. Commun. 3, 1121 (2012).

    Article  ADS  PubMed  Google Scholar 

  46. Bowen, A. M. et al. Ribosomal protein uS19 mutants reveal its role in coordinating ribosome structure and function. Translation 3, e1117703 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Lawson, M. R. et al. Mechanisms that ensure speed and fidelity in eukaryotic translation termination. Science 373, 876–882 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  48. Gromadski, K. B. et al. Kinetics of the Interactions between yeast elongation factors 1 A and 1Bα, guanine nucleotides, and aminoacyl-tRNA. J. Biol. Chem. 282, 35629–35637 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Shapiro, R. & Vallee, B. L. Site-directed mutagenesis of histidine-13 and histidine-114 of human angiogenin. Alanine derivatives inhibit angiogenin-induced angiogenesis. Biochemistry 28, 7401–7408 (1989).

    Article  CAS  PubMed  Google Scholar 

  50. Kirby, J. et al. Lack of unique neuropathology in amyotrophic lateral sclerosis associated with p.K54E angiogenin (ANG) mutation. Neuropathol. Appl. Neurobiol. 39, 562–571 (2013).

    Article  CAS  PubMed  Google Scholar 

  51. Shao, S. et al. Decoding mammalian ribosome-mRNA states by translational GTPase complexes. Cell 167, 1229–1240 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Rybak, S. M. & Vallee, B. L. Base cleavage specificity of angiogenin with Saccharomyces cerevisiae and Escherichia coli 5S RNAs. Biochemistry 27, 2288–2294 (1988).

    Article  CAS  PubMed  Google Scholar 

  53. Loveland, A. B., Demo, G. & Korostelev, A. A. Cryo-EM of elongating ribosome with EF-Tu•GTP elucidates tRNA proofreading. Nature 584, 640–645 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  54. Blázquez, M., Fominaya, J. M. & Hofsteenge, J. Oxidation of sulfhydryl groups of ribonuclease inhibitor in epithelial cells is sufficient for its intracellular degradation. J. Biol. Chem. 271, 18638–18642 (1996).

    Article  PubMed  Google Scholar 

  55. Czech, A., Wende, S., Mörl, M., Pan, T. & Ignatova, Z. Reversible and rapid transfer-RNA deactivation as a mechanism of translational repression in stress. PLoS Genet. 9, e1003767 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Costa, B. et al. Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids. Proc. Natl Acad. Sci. USA 120, e2216330120 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Chen, X. & Wolin, S. L. Transfer RNA halves are found as nicked tRNAs in cells: evidence that nicked tRNAs regulate expression of an RNA repair operon. RNA 29, 620–629 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Akiyama, Y. et al. RTCB complex regulates stress-induced tRNA cleavage. Int. J. Mol. Sci. 23, 13100 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Drino, A. et al. Identification of RNA helicases with unwinding activity on angiogenin-processed tRNAs. Nucleic Acids Res. 51, 1326–1352 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Saikia, M. et al. Angiogenin-cleaved tRNA halves interact with cytochrome c, protecting cells from apoptosis during osmotic stress. Mol. Cell. Biol. 34, 2450–2463 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Chatzileontiadou, D. S. M. et al. The ammonium sulfate inhibition of human angiogenin. FEBS Lett. 590, 3005–3018 (2016).

    Article  CAS  PubMed  Google Scholar 

  62. Sievers, F. et al. Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Madeira, F. et al. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res. 50, W276–W279 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Kurachi, K., Davie, E. W., Strydom, D. J., Riordan, J. F. & Vallee, B. L. Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor. Biochemistry 24, 5494–5499 (1985).

    Article  CAS  PubMed  Google Scholar 

  65. Yoon, J. M., Kim, S. H., Kwon, O. B., Han, S. H. & Kim, B. K. High level expression of soluble angiogenin in Escherichia coli. IUBMB Life 47, 267–273 (1999).

    Article  CAS  Google Scholar 

  66. Holloway, D. E., Hares, M. C., Shapiro, R., Subramanian, V. & Acharya, K. R. High-level expression of three members of the murine angiogenin family in Escherichia coli and purification of the recombinant proteins. Protein Expr. Purif. 22, 307–317 (2001).

    Article  CAS  PubMed  Google Scholar 

  67. Shapiro, R. et al. Expression of Met-(−1) angiogenin in Escherichia coli: conversion to the authentic. Anal. Biochem. 175, 450–461 (1988).

    Article  CAS  PubMed  Google Scholar 

  68. Abeyrathne, P. D., Koh, C. S., Grant, T., Grigorieff, N. & Korostelev, A. A. Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome. eLife 5, e14874 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  69. Walker, S. E. & Fredrick, K. Preparation and evaluation of acylated tRNAs. Methods 44, 81–86 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Eyler, D. E. & Green, R. Distinct response of yeast ribosomes to a miscoding event during translation. RNA 17, 925–932 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Skogerson, L. & Engelhardt, D. Dissimilarity in protein chain elongation factor requirements between yeast and rat liver ribosomes. J. Biol. Chem. 252, 1471–1475 (1977).

    Article  CAS  PubMed  Google Scholar 

  72. Loveland, A. B. et al. Ribosome inhibition by C9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM. Nat. Commun. 13, 2776 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  73. England, C. G., Ehlerding, E. B. & Cai, W. NanoLuc: a small luciferase is brightening up the field of bioluminescence. Bioconjug. Chem. 27, 1175–1187 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Susorov, D., Egri, S. & Korostelev, A. A. Termi-Luc: a versatile assay to monitor full-protein release from ribosomes. RNA https://doi.org/10.1261/rna.076588.120 (2020).

  75. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  76. Svidritskiy, E., Demo, G., Loveland, A. B., Xu, C. & Korostelev, A. A. Extensive ribosome and RF2 rearrangements during translation termination. eLife 8, e46850 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  77. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  PubMed  Google Scholar 

  78. Grant, T., Rohou, A. & Grigorieff, N. cisTEM, user-friendly software for single-particle image processing. eLife 7, e35383 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  79. Lyumkis, D., Brilot, A. F., Theobald, D. L. & Grigorieff, N. Likelihood-based classification of cryo-EM images using FREALIGN. J. Struct. Biol. 183, 377–388 (2013).

    Article  CAS  PubMed  Google Scholar 

  80. Shanmuganathan, V. et al. Structural and mutational analysis of the ribosome-arresting human XBP1u. eLife 8, e46267 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  81. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

    Article  CAS  PubMed  Google Scholar 

  82. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  83. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  ADS  PubMed  Google Scholar 

  84. Shi, H. & Moore, P. B. The crystal structure of yeast phenylalanine tRNA at 1.93 Å resolution: a classic structure revisited. RNA 6, 1091–1105 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Varadi, M. et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 50, D439–D444 (2022).

    Article  CAS  PubMed  Google Scholar 

  86. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  87. Korostelev, A., Bertram, R. & Chapman, M. S. Simulated-annealing real-space refinement as a tool in model building. Acta Crystallogr. D 58, 761–767 (2002).

    Article  ADS  PubMed  Google Scholar 

  88. Chapman, M. S. Restrained real-space macromolecular atomic refinement using a new resolution-dependent electron-density function. Acta Crystallogr. A 51, 69–80 (1995).

    Article  ADS  Google Scholar 

  89. Zhou, G., Wang, J., Blanc, E. & Chapman, M. S. Determination of the relative precision of atoms in a macromolecular structure. Acta Crystallogr. D 54, 391–399 (1998).

    Article  ADS  CAS  PubMed  Google Scholar 

  90. Adams, P. D. et al. The Phenix software for automated determination of macromolecular structures. Methods 55, 94–106 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).

    Article  ADS  CAS  Google Scholar 

  92. DeLano, W. L. The PyMOL molecular graphics system (DeLano Scientific, 2002).

  93. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  94. Ohgane, K. & Yoshioka, H. Quantification of gel bands by an Image J macro, band/peak quantification tool. protocols.io, https://doi.org/10.17504/protocols.io.7vghn3w (2019).

  95. Chan, P. P. & Lowe, T. M. GtRNAdb: a database of transfer RNA genes detected in genomic sequence. Nucleic Acids Res. 37, D93–D97 (2009).

    Article  CAS  PubMed  Google Scholar 

  96. Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank C. Xu, J.-Y. Chang and C. Ouch for data collection at the cryo-EM facility at UMass Medical School; E. Sholi for help with protein purification; D. Conte Jr for comments on the manuscript; and the members of the Jacobson and Korostelev laboratories for discussions. This study was supported by grants from the US National Institutes of Health 1R35GM122468 to A.J. and R35 GM127094 to A.A.K.

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization: A.B.L. and A.A.K. Methodology: A.B.L., A.J. and A.A.K. Validation: A.B.L. and A.A.K. Investigation: A.B.L. and R.G. Resources: A.B.L., R.G., C.S.K., A.J. and A.A.K. Writing—original draft: A.B.L and A.A.K. Writing—review and editing: A.B.L., C.S.K., A.J. and A.A.K. Visualization: A.B.L. Supervision. A.A.K. Funding acquisition: A.A.K.

Corresponding authors

Correspondence to Anna B. Loveland or Andrei A. Korostelev.

Ethics declarations

Competing interests

A.J. is co-founder, director and consultant for PTC Therapeutics. The other authors declare no competing interests.

Peer review

Peer review information

Nature thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Cryo-EM characterization of angiogenin interactions with the ribosome.

(a) Example of hand-picked ribosome particles (red circles) on one of the higher contrast micrographs (n = 600) containing rabbit reticulocyte lysates treated with angiogenin. Representative micrograph had a defocus of −1.4 µm. Notice low contrast of ribosomes among concentrated lysates. (b) Examples of automatic particle picking of ribosome in cisTEM (red circles) on a micrograph containing in vitro assembled 80S–angiogenin(H13A) ribosome complexes. Micrograph was taken on same microscope and has similar defocus and CTF fitting resolution criteria to micrograph in (a). (c) Maximum-likelihood classification in Frealign of a dataset of in vitro assembled 80S ribosome complexes with angiogenin reveals angiogenin bound to the A site of ribosomes in the non-rotated (classical) state. (d) Masked, Fourier shell correlation curve as a function of inverse resolution for the map derived from 45,850 particles shown in Extended Data Fig. 1c (FSC_part from Frealign v9) (80S–angiogenin, black) or for the map from rabbit reticulocyte lysates derived from 1,960 particles shown in Fig. 1d,e (RRL–angiogenin, purple). (e-j) Examples of interactions of angiogenin with the A-site. Model of 80S–angiogenin is shown with the 2.8 Å cryo-EM map, sharpened by a B-factor of −50 Å2 and shown as mesh at σ levels as noted in individual legends. (e) Interaction of angiogenin with H69 of 28S rRNA. Mesh is shown at 2.75 σ. (f) Interaction of angiogenin with 18S rRNA. Mesh is shown at 2.75 σ. (g) Binding of angiogenin near the universally conserved decoding centre residues of H69 (28S rRNA) and h44 (18S rRNA). Mesh for mRNA is shown at 3 σ; for angiogenin and h44, at 4 σ; and for H69 and G626 at 5 σ. mRNA is green for well modelled residues, grey for disordered residues. (h) Interaction of angiogenin with the A-site codon (mRNA is green for well modelled residues, grey for disordered residues). Mesh is shown at 3.5 σ both mRNA and angiogenin. (i) Interaction of angiogenin with P-site tRNA. Mesh is shown at 3.75 σ. (j) Interactions of angiogenin with the first nucleotide of the A-site codon adjacent to the P-tRNA anticodon and codon interaction. Mesh is shown at 4 σ.

Extended Data Fig. 2 Angiogenin cleaves various tRNAs but not isolated stem loops.

(a) Rigid-body docking of in vitro assembled 80S–angiogenin–Ala-tRNAAla–eEF1A–GDPCP model into the RRL–angiogenin cryo-EM map (grey mesh at 6 σ for most of complex, but due to sub-stoichiometry, blue mesh at 4 σ for ternary complex) shows good agreement of in vitro model with the complex visualized directly in rabbit reticulocyte lysates. The density shows that the C-terminal residues of angiogenin form an extended beta strand in lysates and that the ribosome and ternary complex position the anticodon of tRNA near the active site of angiogenin in lysates. (b) Activity of 10 nM angiogenin alone or together with the 80S ribosome complex (including model mRNA encoding phenylalanine) was checked against total rabbit tRNA purified from rabbit reticulocyte lysates. After 100 min, cleavage reactions were separated by denaturing 15% Urea-PAGE gels stained with SYBR Gold (top) then later detected via Northern blotting (bottom) for the 5′ end of three different rabbit tRNA as noted (see Methods). tRNA fragments of all three tRNAs tested increase in the presence of the 80S–angiogenin complex and are prevented by RNasin (n = 2 independent experiments). (c) Activity of angiogenin alone or together with the 80S ribosome complex (including tRNAfMet and model mRNA encoding phenylalanine in A site) was checked using in vitro translated yeast tRNAAla, an extended 33-mer hairpin including the yeast tRNAAla anticodon stem loop, or a 17-mer including only the yeast tRNAAla anticodon stem loop (see Extended Data Fig. 2d). At the specified time (in minutes), cleavage reactions were separated by denaturing 15% Urea-PAGE gels stained with SYBR Gold. Full-length tRNAAla is depleted over time while fragments arise in the presence of the 80S–angiogenin complex, but the 33-mer and 17-mer are not depleted suggesting tRNA is cleaved more effectively than a simple hairpin. Weak tRNA-fragment-sized bands appearing in 33-mer and 17-mer lanes likely arise from tRNAfMet, which is present in the 80S complex for positioning in the P-site. (n = 2 independent experiments). (d) Diagrams depicting the secondary structure of in vitro translated yeast tRNAAla, the extended 33-mer hairpin including the yeast tRNAAla anticodon stem loop, or the 17-mer hairpin used in Extended Data Fig. 2c. Larger, bold residues are the anticodon of tRNAAla. (e) Quantification of full length tRNA and hairpins from Extended Data Fig. 2c. The amount of full-length tRNA decreased by 50–66% from 1 to 100 min while the amount of hairpins stays constant. (bar shows mean of n = 2 independent experiments).

Extended Data Fig. 3 Purification and characterization of WT, H13A and K54E angiogenin.

(a) SDS PAGE showing inclusion-body expressed, refolded and purified WT, H13A and K54E angiogenin (“refolded” Angiogenin see Methods, Preparation of Angiogenin) (n = 1). (b) 200 nM WT refolded angiogenin co-pellets with purified ribosomes more efficiently than angiogenin(K54E). Bovine pancreatic RNase A does not co-pellet with ribosome under these conditions (n = 1). (c) RRL translation of nanoluciferase-encoding mRNA is inhibited by 90 nM refolded WT but not H13A or K54E angiogenin. These data, excluding RNasin controls, also appear in Fig. 2k. (mean +/− s.d., n = 2 for mutants or 6 for controls, independent experiments). (d) Refolded WT angiogenin and angiogenin(K54E) but not angiogenin(H13A) exhibit weak RNase activity against total yeast tRNA in the absence of ribosomes, at high enzyme concentration and high tRNA concentration3,44 (50 and 300 times higher, respectively, than in the ribosome-activated cleavage assay), indicating that K54E mutation does not affect the basal catalytic activity (Methods) (mean +/− s.e., n = 2 independent experiments). (e-f) SDS PAGE showing recombinant, soluble WT angiogenin (e) and angiogenin(H13A) (f) (“soluble” angiogenin, Methods, Preparation of Angiogenin) in comparison with 200 ng of R&D angiogenin. * is a contaminant in soluble angiogenin(H13A) prep (n = 1). (g) Western blotting of soluble angiogenin(H13A) and R&D angiogenin shows similar amounts are loaded (n = 1). (h) Comparison of tRNAAla cleavage by R&D vs. soluble WT and H13A angiogenin (10 nM). A ternary complex containing Ala-tRNAAla, eEF1A and GDPCP was treated with angiogenin in the presence or absence of the 80S ribosome complex with UUC-containing mRNA and tRNAfMet. After 0 or 10 min, cleavage reactions were separated by denaturing 15% Urea-PAGE gels and stained with SYBR Gold. R&D and soluble WT angiogenin exhibit the same activity, while angiogenin(H13A) is inactive. (mean, n = 2 independent experiments). (i) Translation of nanoluciferase-encoding mRNA is inhibited by soluble WT angiogenin (similarly to R&D angiogenin shown in Fig. 1a), but not by the soluble angiogenin(H13A) (RLU: relative light units). Example trace is shown. (j) Apparent rate of translation, measured as RLU/s (see Extended Data Fig. 3i), in the presence of soluble WT angiogenin or angiogenin(H13A), (mean +/− s.d., n = 3 independent experiments except buffer control which was duplicated for n = 6).

Extended Data Fig. 4 Effects of ribosome complex composition on angiogenin activation.

(a) Production of tRNAAla fragments by angiogenin is stimulated by an elongation-like 80S ribosome complex more efficiently than by 40S or 60S subunits. A ternary complex containing Ala-tRNAAla, eEF1A and GDPCP was supplemented to 80S ribosome complexes assembled using 40S, 60S, leaderless mRNA placing phenylalanine UUC codon in A site, tRNAfMet complementary to the P-site, and/or angiogenin. After 10 min, cleavage reactions were separated by denaturing 15% Urea-PAGE gels stained with SYBR Gold (top) then later detected via Northern blotting for the 5′ end of yeast tRNAAla (bottom), n = 2 independent experiments. (b) Same reactions as in (a) run separately then probed for the 3′ end of yeast tRNAAla, n = 2 independent experiments. (c) Close-up of lane 14 from (a) showing SYBR Gold and 5′ Northern side-by-side. Two 5′ tRNA fragments are apparent with the more intense one running above the 30 nt marker. (d) Close-up of lane 14 from (b) showing SYBR Gold and 3′ Northern side-by-side. 3′ tRNA fragment runs close to the 40 nt marker. (e) Secondary structure diagram of tRNAAla with the anticodon shown in orange font. Triangles indicate potential cleavage locations in the anticodon stem based on preference of angiogenin to cleaved at pyrimidine-adenine dinucleotides52. (f) Cleavage of tRNAAla by angiogenin is stimulated by multiple 80S ribosome complexes including ones lacking mRNA, lacking P-site tRNA, and ones encoding amino acids phenylalanine (F, UUC) or lysine (K, AAA), or a stop codon (X, UAA) as well as by truncated mRNA (1, single uridine in A site). A ternary complex Ala-tRNAAla, eEF1A and GDPCP was supplemented to ribosome complexes assembled using 40S, 60S, mRNAs as specified, tRNAfMet complementary to the P-site, and/or angiogenin, and after the indicated time (in minutes) cleavage reactions were stopped then separated by denaturing 15% Urea-PAGE gels stained with SYBR Gold (top) then later detected via Northern blotting (bottom) for the 5′ end of yeast tRNAAla. (mean, n = 2 independent experiments).

Extended Data Fig. 5 Cryo-EM of the 80S–angiogenin and 80S–angiogenin(H13A) complexes with Ala-tRNAAla–eEF1A–GDPCP ternary complex.

(a) Maximum-likelihood classification in Frealign of a dataset of 80S ribosome with angiogenin and ternary complex of Ala-tRNAAla–eEF1A–GDPCP reveals classes with angiogenin bound to the ribosomal A site as tRNA is presented to the angiogenin active site. (b) Masked, Fourier shell correlation curves (FSC) as a function of inverse resolution for the cryo-EM map described in Extended Data Fig. 5a. (c) Maximum-likelihood classification in Frealign of a dataset collected from 80S ribosomes with recombinantly expressed angiogenin(H13A) and ternary complex of Ala-tRNAAla–eEF1A–GDPCP. (d) Masked, Fourier shell correlation curves (FSC) as a function of inverse resolution for the cryo-EM maps described in Extended Data Fig. 5c. (e) Half reactions for assembling the cryo-EM complexes in Extended Data Fig. 5a (Half reaction 1: 80S ribosome complexes with angiogenin; Half reaction 2: ternary complex of Ala-tRNAAla–eEF1A–GDPCP as described in Methods) were mixed in test tubes held on wet ice. After the indicated time, the reactions were quenched with 8 M Urea loading buffer. For the 0-s time point, Half reaction 1 and Half reaction 2 were added directly to 8 M Urea loading buffer. The reactions were separated on a 15% Urea-PAGE gel to visualize full length and newly cleaved tRNA. tRNA is cleaved nearly maximally by 25 s on ice under these conditions, which mimic the conditions used to prepare cryo-EM grids (n = 1). (f) Half reactions for assembling the cryo-EM complexes in Extended Data Fig. 5c (Half reaction 1: 80S ribosome complexes with recombinant angiogenin(H13A); Half reaction 2: ternary complex of Ala-tRNAAla–eEF1A–GDPCP as described in Methods) were mixed in test tubes at 30 °C. After the indicated time, the reactions were quenched with 8 M Urea loading buffer and separated showing lack of tRNA cleavage under these conditions, which mimic the conditions used to prepare cryo-EM grids (300 s) (n = 1).

Extended Data Fig. 6 Cryo-EM density maps of the 80S–angiogenin(WT) and angiogenin(H13A) complexes formed with Ala-tRNAAla–eEF1A–GDPCP.

(a) 3.7-Å cryo-EM map of 80S–angiogenin(WT)–Ala-tRNAAla–eEF1A–GDPCP has density consistent with GDPCP (cyan) bound to eEF1A (blue). GDPCP-bound bacterial EF-Tu (cyan) (PDB: 5UYM) was aligned via the homologous domain 1 to domain 1 of eEF1A. Mesh is shown at 2.75 σ. (b). The 2.8-Å cryo-EM map of 80S–angiogenin (WT), sharpened by a B-factor of −50 Å2, has well-resolved density for the active site residue H13. Mesh is shown at 4 σ. (c) The 2.8-Å cryo-EM map of 80S–angiogenin(H13A) (no sharpening) has well-resolved density for the active site of angiogenin supporting mutation of histidine 13 to alanine (H13A) (mesh). The H13A map was rigid-body fitted with our 80S–angiogenin model. Mesh is shown at 4 σ. (d-f) Comparison of density for tRNAAla in the 3.7-Å cryo-EM map of 80S–angiogenin(WT)–Ala-tRNAAla–eEF1A–GDPCP (d), 3.1-Å cryo-EM map of 80S–angiogenin(WT)–tRNAAla (e) or 3.9-Å cryo-EM map of 80S–angiogenin(H13A)–tRNAAla (f) show that the anticodon stem–loop (ASL) of tRNA is strongest in the H13A map with the catalytically inactive angiogenin. The cryo-EM maps (grey mesh) are shown without sharpening at 4 σ. (g-i) Comparison of cryo-EM density filtered to 5 Å in the cryo-EM maps of 80S–angiogenin(WT)–Ala-tRNAAla–eEF1A–GDPCP (g), 80S–angiogenin(WT)–tRNAAla (h) or 80S–angiogenin(H13A)–tRNAAla (i) further supports that the ASL of tRNA is stronger in the H13A map with catalytically inactive angiogenin. (j-l) The cryo-EM maps of 80S–angiogenin(WT)–Ala-tRNAAla–eEF1A–GDPCP (j), 80S–angiogenin(WT)–tRNAAla (k) or 80S–angiogenin(H13A)–tRNAAla (l) show an L-shaped density consistent with tRNA interacting with the P-stalk via the tRNA elbow. The cryo-EM maps were each low-pass filtered to 5 Å and shown at 3 σ. (m) Cryo-EM map of 80S–angiogenin–Ala-tRNAAla–eEF1A–GDPCP shows density for eEF1A (blue) bound to the shoulder of the 40S (gold). The cryo-EM map was low-pass filtered to 5 Å and is shown at 2.7 σ. (n) The cryo-EM map of 80S–angiogenin–Ala-tRNAAla–eEF1A–GDPCP has an unidentified low-resolution density (question mark) next to the anticodon arm of Ala-tRNAAla (green) and the P-stalk (uL11, light blue is labelled for reference), which may correspond to a dynamic element of the P-stalk. The cryo-EM map was low-pass filtered to 5 Å and is shown at 2.5 σ.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

Supplementary information

Supplementary Information

Supplementary Discussion, full descriptions for Supplementary Videos 1 and 2, Supplementary Fig. 1 and Supplementary References.

Reporting Summary

Supplementary Video 1

Cryo-EM maps and rigid-body fits of the subclasses of 80S–angiogenin–tRNAAla show different positions of tRNA (green) and P stalk (cyan, top left corner) relative to angiogenin (magenta). This flexible tethering of the tRNA may allow cleavage at different anticodon positions on various tRNA species. The cryo-EM maps were low-pass filtered to 6 Å and are shown at 2.5σ.

Supplementary Video 2

Cryo-EM maps and rigid-body fits of the subclasses of 80S–angiogenin–tRNAAla show different positions of tRNA (green) and P stalk (cyan, top left corner) relative to the 60S subunit’s GTPase activation centre. The cryo-EM maps were low-pass filtered to 6 Å and are shown at 2.5σ.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Loveland, A.B., Koh, C.S., Ganesan, R. et al. Structural mechanism of angiogenin activation by the ribosome. Nature 630, 769–776 (2024). https://doi.org/10.1038/s41586-024-07508-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-024-07508-8

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing