The ccc proline codon preceding a stop codon modulates translation termination in eukaryotes depending on the molecular context

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription or Fee Access

Abstract

In bacteria, glycine and proline codons located upstream of stop codons suppress translation termination. However, the effects of those codons in eukaryotes has not been systematically investigated. In this study, we demonstrate that preceding stop codon CCC codon of proline suppresses translation termination in eukaryotes during the synthesis of long protein. Conversely, during synthesis of short peptide, a proline codon in this position stimulates the formation of termination complexes. Furthermore, we investigated the role of poly(A)-binding protein (PABP), a key regulator of eukaryotic translation termination associated with the poly(A) tail of mRNA, in modulation of translation termination by the 5ʹ context of stop codons. Our findings reveal that during the synthesis of short peptides, PABP reduces dependence of translation termination on the 5ʹ stop codon contexts and promotes translation termination independently of the 5ʹ stop codon context during the synthesis of long proteins.

About the authors

N. S. Biziaev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Moscow, 119991 Russia

A. V. Shuvalov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Moscow, 119991 Russia; Moscow, 119991 Russia

E. Z. Alkalaeva

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: alkalaeva@eimb.ru
Moscow, 119991 Russia; Moscow, 119991 Russia

References

  1. Alkalaeva E.Z., Pisarev A.V., Frolova L.Y., Kisselev L.L., Pestova T.V. (2006) In vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3. Cell. 125, 1125–1136.
  2. Hellen C.U.T. (2018) Translation termination and ribosome recycling in eukaryotes. Cold Spring Harbor Persp. Biol. 10, I. 10. 1–18.
  3. Egorova T., Biziaev N., Shuvalov A., Sokolova E., Mukba S., Evmenov K., Zotova M., Kushchenko A., Shuvalova E., Alkalaeva E. (2021) eIF3j facilitates loading of release factors into the ribosome. Nucl. Acids Res. 49, 11181–11196.
  4. Ivanov A., Mikhailova T., Eliseev B., Yeramala L., Sokolova E., Susorov D., Shuvalov A., Schaffitzel C., Alkalaeva E. (2016) PABP enhances release factor recruitment and stop codon recognition during translation termination. Nucl. Acids Res. 44, 7766–7776.
  5. Biziaev N., Shuvalov A., Salman A., Egorova T., Shuvalova E., Alkalaeva E. (2024) The impact of mRNA poly(A) tail length on eukaryotic translation stages. Nucl. Acids Res. 52, 7792–7808.
  6. Wu C., Roy B., He F., Yan K., Jacobson A. (2020) Poly(A)-binding protein regulates the efficiency of translation termination. Cell Rep. 33, 108399.
  7. Ivanov P.V., Gehring N.H., Kunz J.B., Hentze M.W., Kulozik A.E. (2008) Interactions between UPF1, eRFs, PABP and the exon junction complex suggest an integrated model for mammalian NMD pathways. EMBO J. 27, 736–747.
  8. Cosson B., Berkova N., Couturier A., Chabelskaya S., Philippe M., Zhouravleva G. (2002) Poly(A)-binding protein and eRF3 are associated in vivo in human and Xenopus cells. Biol. Cell. 94, 205–216.
  9. Hoshino S., Imai M., Kobayashi T., Uchida N., Katada T. (1999) The eukaryotic polypeptide chain releasing factor (eRF3/GSPT) carrying the translation termination signal to the 3ʹ-poly(A) tail of mRNA. J. Biol. Chem. 274, 16677–16680.
  10. Uchida N., Hoshino S., Imataka H., Sonenberg N., Katada T. (2002) A novel role of the mammalian GSPT/eRF3 associating with poly(A)-binding protein in cap/poly(A)-dependent translation. J. Biol. Chem. 277, 50286–50292.
  11. Бизяев Н.С., Егорова Т.В., Алкалаева Е.З. (2022) Динамика структуры мРНК эукариот в ходе трансляции. Молекуляр. биология. 56, 451–464.
  12. Lima S.A., Chipman L.B., Nicholson A.L., Chen Y.H., Yee B.A., Yeo G.W., Coller J., Pasquinelli A.E. (2017) Short poly(A) tails are a conserved feature of highly expressed genes. Nat. Struct. Mol. Biol. 24, 1057–1063.
  13. Beier H., Grimm M. (2001) Misreading of termination codons in eukaryotes by natural nonsense suppressor tRNAs. Nucl. Acids Res. 29, 4767–4782.
  14. Bertram G., Innes S., Minella O., Richardson J., Stansfield I. (2001) Endless possibilities: translation termination and stop codon recognition. Microbiology. 147, 255–269.
  15. Roy B., Leszyk J.D., Mangus D.A., Jacobson A. (2015) Nonsense suppression by near-cognate tRNAs employs alternative base pairing at codon positions 1 and 3. Proc. Natl. Acad. Sci. USA. 112, 3038–3043.
  16. Vallabhaneni H., Fan-Minogue H., Bedwell D.M., Farabaugh P.J. (2009) Connection between stop codon reassignment and frequent use of shifty stop frameshifting. RNA. 15, 889–897.
  17. Kurian L., Palanimurugan R., Gödderz D., Dohmen R.J. (2011) Polyamine sensing by nascent ornithine decarboxylase antizyme stimulates decoding of its mRNA. Nature. 477, 490–494.
  18. Amrani N., Sachs M.S., Jacobson A. (2006) Early nonsense: mRNA decay solves a translational problem. Nat. Rev. Mol. Cell. Biol. 7, 415–425.
  19. Celik A., Kervestin S., Jacobson A. (2015) NMD: at the crossroads between translation termination and ribosome recycling. Biochimie. 114, 2–9.
  20. Raimondeau E., Bufton J.C., Schaffitzel C. (2018) New insights into the interplay between the translation machinery and nonsense-mediated mRNA decay factors. Biochem. Soc. Trans. 46, 503–512.
  21. Embree C.M., Abu-Alhasan R., Singh G. (2022) Features and factors that dictate if terminating ribosomes cause or counteract nonsense-mediated mRNA decay. J. Biol. Chem. 298, 102592.
  22. Соколова Е.Е., Власов П.К., Егорова Т.В., Шувалов А.В., Алкалаева Е.З. (2020) Влияние А/G-состава 3ʹ-контекстов стоп-кодонов на терминацию трансляции у эукариот. Молекуляр. биология. 54, 837–848.
  23. Cridge A.G., Crowe-McAuliffe C., Mathew S.F., Tate W.P. (2018) Eukaryotic translational termination efficiency is influenced by the 3′ nucleotides within the ribosomal mRNA channel. Nucl. Acids Res. 46, 1927–1944.
  24. Björnsson A., Mottagui-Tabar S., Isaksson L.A. (1996) Structure of the C-terminal end of the nascent peptide influences translation termination. EMBO J. 15, 1696–1704.
  25. Mottagui-Tabar S., Tuite M.F., Isaksson L.A. (1998) The influence of 5’ codon context on translation termination in Saccharomyces cerevisiae. Eur. J. Biochem. 257, 249–254.
  26. Tork S., Hatin I., Rousset J.P., Fabret C. (2004) The major 5ʹ determinant in stop codon read-through involves two adjacent adenines. Nucl. Acids Res. 32, 415–421.
  27. Wangen J.R., Green R. (2020) Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides. ELife. 9, 1–29.
  28. Dabrowski M., Bukowy-Bieryllo Z., Zietkiewicz E. (2015) Translational readthrough potential of natural termination codons in eucaryotes — The impact of RNA sequence. RNA Biol. 12, 950–958.
  29. Cassan M., Rousset J.P. (2001) UAG readthrough in mammalian cells: effect of upstream and downstream stop codon contexts reveal different signals. BMC Mol. Biol. 2, article number 3, 1–8.
  30. Bonetti B., Fu L., Moon J., Bedwell D.M. (1995) The efficiency of translation termination is determined by a synergistic interplay between upstream and downstream sequences in Saccharomyces cerevisiae. J. Mol. Biol. 251, 334–345.
  31. Loughran G., Chou M.Y., Ivanov I.P., Jungreis I., Kellis M., Kiran A.M., Baranov P.V., Atkins J.F. (2014) Evidence of efficient stop codon readthrough in four mammalian genes. Nucl. Acids Res. 42, 8928–8938.
  32. Williams I., Richardson J., Starkey A., Stansfield I. (2004) Genome-wide prediction of stop codon readthrough during translation in the yeast Saccharomyces cerevisiae. Nucl. Acids Res. 32, 6605–6616.
  33. Bohlen J., Harbrecht L., Blanco S., Clemm von Hohenberg K., Fenzl K., Kramer G., Bukau B., Teleman A.A. (2020) DENR promotes translation reinitiation via ribosome recycling to drive expression of oncogenes including ATF4. Nat. Commun. 11, 4676, 1–15.
  34. Young D.J., Meydan S., Guydosh N.R. (2021) 40S ribosome profiling reveals distinct roles for Tma20/Tma22 (MCT-1/DENR) and Tma64 (eIF2D) in 40S subunit recycling. Nat. Commun. 12, 2976, 1–16.
  35. Young D.J., Guydosh N.R. (2022) Rebirth of the translational machinery: the importance of recycling ribosomes. BioEssays. 44, 2100269, 1–16.
  36. Kolakada D., Fu R., Biziaev N., Shuvalov A., Lore M., Campbell A.E., Cortázar M.A., Sajek M.P., Hesselberth J.R., Mukherjee N., Alkalaeva E., Coban-Akdemir Z.H., Jagannathan S. (2025) Systematic analysis of nonsense variants uncovers peptide release rate as a novel modifier of nonsense-mediated mRNA decay. Cell Genomics, 100882.
  37. Pierson W.E., Hoffer E.D., Keedy H.E., Simms C.L., Dunham C.M., Zaher H.S. (2016) Uniformity of peptide release is maintained by methylation of release factors. Cell Rep. 17, 11–18.
  38. Meydan S., Guydosh N.R. (2020) Disome and trisome profiling reveal genome-wide targets of ribosome quality control. Mol. Cell. 79, 588–602.e6.
  39. Schuller A.P., Wu C.C.C., Dever T.E., Buskirk A.R., Green R. (2017) eIF5A functions globally in translation elongation and termination. Mol. Cell. 66, 194–205.e5.
  40. Gutierrez E., Shin B.-S., Woolstenhulme C.J., Kim J.-R., Saini P., Buskirk A.R., Dever T.E. (2013) eIF5A promotes translation of polyproline motifs. Mol. Cell. 51, 35–45.
  41. Schuller A.P., Green R. (2018) Roadblocks and resolutions in eukaryotic translation. Nat. Rev. Mol. Cell Biol. 19, 526–541.
  42. Doerfel L.K., Wohlgemuth I., Kothe C., Peske F., Urlaub H., Rodnina M.V. (2013) EF-P is essential for rapid synthesis of proteins containing consecutive proline residues. Science. 339, 85–88.
  43. Ude S., Lassak J., Starosta A.L., Kraxenberger T., Wilson D.N., Jung K. (2013) Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches. Science. 339, 82–85.
  44. Pavlov M.Y., Watts R.E., Tan Z., Cornish V.W., Ehrenberg M., Forster A.C. (2009) Slow peptide bond formation by proline and other N-alkylamino acids in translation. Proc. Natl. Acad. Sci. USA. 106, 50–54.
  45. Wohlgemuth I., Brenner S., Beringer M., Rodnina M.V. (2008) Modulation of the rate of peptidyl transfer on the ribosome by the nature of substrates. J. Biol. Chem. 283, 32229–32235.
  46. Коростелев А.A. (2021) Различия и сходство процессов терминации трансляции и спасения рибосомы в бактериальных клетках и в митохондриях и цитоплазме эукариотических клеток. Биохимия. 86, 1328–1344.
  47. Donnelly M.L.L., Luke G., Mehrotra A., Li X., Hughes L.E., Gani D., Ryan M.D. (2001) Analysis of the aphthovirus 2A/2B polyprotein “cleavage” mechanism indicates not a proteolytic reaction, but a novel translational effect: а putative ribosomal “skip.” J. Gen. Virol. 82, 1013–1025.
  48. Sharma P., Yan F., Doronina V.A., Escuin-Ordinas H., Ryan M.D., Brown J.D. (2012) 2A peptides provide distinct solutions to driving stop-carry on translational recoding. Nucl. Acids Res. 40, 3143–3151.
  49. Ito K., Chiba S. (2013) Arrest peptides: сis-acting modulators of translation. Annu. Rev. Biochem. 82, 171–202.
  50. Shuvalov A., Shuvalova E., Biziaev N., Sokolova E., Evmenov K., Pustogarov N., Arnautova A., Matrosova V., Egorova T., Alkalaeva E. (2021) Nsp1 of SARS-CoV-2 stimulates host translation termination. RNA Biol. 18, sup.2, 1–14.
  51. Shuvalov A., Klishin A., Biziaev N., Shuvalova E., Alkalaeva E. (2024) Functional аctivity of isoform 2 of human eRF1. Internat. J. Mol. Sci. 25, 7997.
  52. Susorov D., Egri S., Korostelev A.A. (2020) Termi-Luc: a versatile assay to monitor full-protein release from ribosomes. RNA. 26, 2044–2050.
  53. Egorova T., Sokolova E., Shuvalova E., Matrosova V., Shuvalov A., Alkalaeva E. (2019) Fluorescent toeprinting to study the dynamics of ribosomal complexes. Methods. 162–163, 54–59.
  54. Shirokikh N.E., Alkalaeva E.Z., Vassilenko K.S., Afonina Z.A., Alekhina O.M., Kisselev L.L., Spirin A.S. (2009) Quantitative analysis of ribosome-mRNA complexes at different translation stages. Nucl. Acids Res. 38. e15.
  55. Holm S. (1979) A simple sequentially rejective multiple test procedure. Scandinavian J. Statistics. 6, 65–70.
  56. Frolova L.Y., Tsivkovskii R.Y., Sivolobova G.F., Oparina N.Y., Serpinsky O.I., Blinov V.M., Tatkov S.I., Kisselev L.L. (1999) Mutations in the highly conserved GGQ motif of class I polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis. RNA. 5, 1014–1020.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download

Copyright (c) 2025 Russian Academy of Sciences