Human eRT1 Translation Regulation

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

Translation termination factor eRF1 is an important cellular protein that plays a key role in translation termination, nonsense-mediated mRNA decay (NMD), and stop-codons readthrough. An amount of eRF1 in the cell influences all these processes. The mechanism of eRF1 translation regulation through an autoregulatory NMD-dependent expression circuit has been described for plants and fungi, but the mechanisms of human eRF1 translation regulation have not yet been studied. Using reporter constructs, we studied the effect of eRF1 mRNA elements on its translation in cell-free translation systems and HEK293 cells. Our data do not support the presence of the NMD-dependent autoregulatory circuit of human eRF1 expression. We found that the 5'-untranslated region (5'-UTR) of eRF1 mRNA and the start codon of the upstream open reading frame (uORF) have the greatest influence on the translation of CDS. According to the DataBase of Transcriptional Start Sites (DBTSS), eRF1 mRNA has a high heterogeneity of transcription start sites and variable length of 5'-UTRs as a consequence. Moreover, the start codon of the eRF1 CDS is located within the known Translation Initiator of Short 5′UTR (TISU), which also stimulates mRNA transcription of genes with high transcription start heterogeneity. We hypothesize that regulation of eRF1 mRNA translation occurs at both the transcriptional and translational levels. At the transcription level, the length of the 5'-UTRs of eRF1 and the number of short open reading frames in it are regulated, which in turn regulate eRF1 production at the translational level.

Full Text

Restricted Access

About the authors

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

Author for correspondence.
Email: alkalaeva@eimb.ru
Russian Federation, Moscow, 119991; Moscow, 119991

A. A. Klishin

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences; Moscow Institute of Physics and Technology

Email: alkalaeva@eimb.ru
Russian Federation, Moscow, 119991; Moscow Region, Dolgoprudny, 141700

N. S. Biziaev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: alkalaeva@eimb.ru
Russian Federation, Moscow, 119991

E. Y. Shuvalova

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
Russian Federation, Moscow, 119991; Moscow, 119991

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; Moscow Institute of Physics and Technology

Email: alkalaeva@eimb.ru
Russian Federation, Moscow, 119991; Moscow, 119991; Moscow Region, Dolgoprudny, 141700

References

  1. Bertram G., Bell H.A., Ritchie D.W., Fullerton G., Stansfield I. (2000) Terminating eukaryote translation: domain 1 of release factor eRF1 functions in stop codon recognition. RNA. 6, 1236–1247.
  2. Frolova L., Seit-Nebi A., Kisselev L. (2002) Highly conserved NIKS tetrapeptide is functionally essential in eukaryotic translation termination factor eRF1. RNA. 8, 129–136.
  3. Bulygin K.N., Khairulina Y.S., Kolosov P.M., Ven'yaminova A.G., Graifer D.M., Vorobjev Y.N., Frolova L.Y., Kisselev L.L., Karpova G.G. (2010) Three distinct peptides from the N domain of translation termination factor eRF1 surround stop codon in the ribosome. RNA. 16, 1902–1914.
  4. Bulygin K.N., Khairulina Y.S., Kolosov P.M., Ven'yaminova A.G., Graifer D.M., Vorobjev Y.N., Frolova L.Y., Karpova G.G. (2011) Adenine and guanine recognition of stop codon is mediated by different N domain conformations of translation termination factor eRF1. Nucl. Acids Res. 39, 7134–7146.
  5. Kryuchkova P., Grishin A., Eliseev B., Karyagina A., Frolova L., Alkalaeva E. (2013) Two-step model of stop codon recognition by eukaryotic release factor eRF1. Nucl. Acids Res. 41, 4573–4586.
  6. Brown A., Shao S., Murray J., Hegde R.S., Ramakrishnan V. (2015) Structural basis for stop codon recognition in eukaryotes. Nature. 524, 493–496.
  7. 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 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis. RNA. 5, 1014–1020.
  8. Zhouravleva G., Frolova L., Le Goff X., Le Guellec R., Inge-Vechtomov S., Kisselev L., Philippe M. (1995) Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. EMBO J. 14, 4065–4072.
  9. Frolova L., Le Goff X., Zhouravleva G., Davydova E., Philippe M., Kisselev L. (1996) Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase. RNA. 2, 334–341.
  10. Stansfield I., Jones K.M., Kushnirov V.V., Dagkesamanskayal A.R., Poznyakovski A., Paushkin S.V., Nierras C.R., Cox B.S., Ter-Avanesyan M.D., Tuite M.F. (1995) The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. EMBO J. 14, 4365–4373.
  11. Paushkin S.V., Kushnirov V.V., Smirnov V.N., Ter-Avanesyan M.D. (1997) Interaction between yeast Sup45p (eRF1) and Sup35p (eRF3) polypeptide chain release factors: implications for prion-dependent regulation. Mol. Cell. Biol. 17, 2798–2805.
  12. Ito K., Ebihara K., Nakamura Y. (1998) The stretch of C-terminal acidic amino acids of translational release factor eRF1 is a primary binding site for eRF3 of fission yeast. RNA. 4, S1355838298971874.
  13. Merkulova T.I., Frolova L.Y., Lazar M., Camonis J., Kisselev L.L. (1999) C-terminal domains of human translation termination factors eRF1 and eRF3 mediate their in vivo interaction. FEBS Lett. 443, 41–47.
  14. Namy O., Hatin I., Rousset J. (2001) Impact of the six nucleotides downstream of the stop codon on translation termination. EMBO Rep. 2, 787–793.
  15. Atkins J.F., Loughran G., Bhatt P.R., Firth A.E., Baranov P.V. (2016) Ribosomal frameshifting and transcriptional slippage: from genetic steganography and cryptography to adventitious use. Nucl. Acids Res. 44, 7007–7078
  16. Rodnina M.V., Korniy N., Klimova M., Karki P., Peng B. Z., Senyushkina T., Belardinelli R., Maracci C., Wohlgemuth I., Samatova E., Peske F. (2020) Translational recoding: canonical translation mechanisms reinterpreted. Nucl. Acids Res. 48, 1056–1067.
  17. Brogna S., Wen J. (2009) Nonsense-mediated mRNA decay (NMD) mechanisms. Nat. Struct. Mol. Biol.16, 107–113.
  18. Шабельская С.В., Журавлева Г.А. (2010) Мутации в гене SUP35 нарушают процесс деградации мРНК, содержащих преждевременные стоп-кодоны. Молекуляр. биология. 44, 51–59.
  19. Schweingruber C., Rufener S.C., Zünd D., Yamashita A., Mühlemann O. (2013) Nonsense-mediated mRNA decay – Mechanisms of substrate mRNA recognition and degradation in mammalian cells. Biochim. Biophys. Acta (BBA) – Gene Regulatory Mechanisms. 1829, 612–623.
  20. Karousis E.D., Gurzeler L.-A., Annibaldis G., Dreos R., Mühlemann O. (2020) Human NMD ensues independently of stable ribosome stalling. Nat. Commun. 11, 4134.
  21. Yang Q., Yu C.-H., Zhao F., Dang Y., Wu C., Xie P., Sachs M.S., Liu Y. (2019) eRF1 mediates codon usage effects on mRNA translation efficiency through premature termination at rare codons. Nucl. Acids Res. 47, 9243–9258.
  22. Wada M., Ito K. (2019) Misdecoding of rare CGA codon by translation termination factors, eRF1/eRF3, suggests novel class of ribosome rescue pathway in S. cerevisiae. FEBS J. 286, 788–802.
  23. Dever T.E., Ivanov I.P., Sachs M.S. (2020) Conserved upstream open reading frame nascent peptides that control translation. Annu. Rev. Genet. 54, 237–264.
  24. Bidou L., Allamand V., Rousset J.-P., Namy O. (2012) Sense from nonsense: therapies for premature stop codon diseases. Trends Mol. Med. 18, 679–688.
  25. Janzen D.M. (2004) The effect of eukaryotic release factor depletion on translation termination in human cell lines. Nucl. Acids Res. 32, 4491–4502.
  26. Freitag J., Ast J., Bölker M. (2012) Cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi. Nature. 485, 522–525.
  27. Schueren F., Lingner T., George R., Hofhuis J., Dickel C., Gärtner J., Thoms S. (2014) Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals. ELife. 3, e03640
  28. Hofhuis J., Schueren F., Nötzel C., Lingner T., Gärtner J., Jahn O., Thoms S. (2016) The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code. Open Biol. 6, 160246.
  29. Svidritskiy E., Demo G., Korostelev A.A. (2018) Mechanism of premature translation termination on a sense codon. J. Biol. Chem. 293, 12472–12479.
  30. Dubourg C., Toutain B., Le Gall J.-Y., Le Treut A., Guenet L. (2003) Promoter analysis of the human translation termination factor 1 gene. Gene. 316, 91–101.
  31. Nyikó T., Auber A., Szabadkai L., Benkovics A., Auth M., Mérai Z., Kerényi Z., Dinnyés A., Nagy F., Silhavy D. (2017) Expression of the eRF1 translation termination factor is controlled by an autoregulatory circuit involving readthrough and nonsense-mediated decay in plants. Nucl. Acids Res. 45, 4174–4188.
  32. Kurilla A., Szőke A., Auber A., Káldi K., Silhavy D. (2020) Expression of the translation termination factor eRF1 is autoregulated by translational readthrough and 3'UTR intron‐mediated NMD in Neurospora crassa. FEBS Lett. 594, 3504–3517.
  33. 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, 804–817.
  34. СоколоваЕ.Е., Власов П.К., Егорова Т.В., Шувалов А.В., Алкалаева Е.З. (2020) Влияние А/G-состава 3'-контекстов стоп-кодонов на терминацию трансляции у эукариот. Молекуляр. биология. 54, 837–848.
  35. Biziaev N., Sokolova E., Yanvarev D.V., Toropygin I.Y., Shuvalov A., Egorova T., Alkalaeva E. (2022) Recognition of 3′ nucleotide context and stop codon readthrough are determined during mRNA translation elongation. J. Biol. Chem. 298, 102133.
  36. Holm S. (1979) A simple sequentially rejective multiple test procedure. Scandinavian J. Statistics. 6, 65–70.
  37. Suzuki A., Kawano S., Mitsuyama T., Suyama M., Kanai Y., Shirahige K., Sasaki H., Tokunaga K., Tsuchihara K., Sugano S., Nakai K., Suzuki Y. (2018) DBTSS/DBKERO for integrated analysis of transcriptional regulation. Nucl. Acids Res. 46, D229–D238.
  38. Akulich K.A., Andreev D.E., Terenin I.M., Smirnova V.V., Anisimova A.S., Makeeva D.S., Arkhipova V.I., Stolboushkina E.A., Garber M.B., Prokofjeva M.M., Spirin P.V., Prassolov V.S., Shatsky I.N., Dmitriev S.E. (2016) Four translation initiation pathways employed by the leaderless mRNA in eukaryotes. Sci. Rep. 6, 37905.
  39. Lin Y., May G.E., Kready H., Nazzaro L., Mao M., Spealman P., Creeger Y., McManus C.J. (2019) Impacts of uORF codon identity and position on translation regulation. Nucl. Acids Res. 47, 9358–9367.
  40. Dever T.E., Ivanov I.P., Hinnebusch A.G. (2023) Translational regulation by uORFs and start codon selection stringency. Genes Dev. 37, 474–489.
  41. Starck S.R., Tsai J.C., Chen K., Shodiya M., Wang L., Yahiro K., Martins-Green M., Shastri N., Walter P. (2016) Translation from the 5′ untranslated region shapes the integrated stress response. Science. 351, aad3867
  42. Akulich K.A., Sinitcyn P.G., Makeeva D.S., Andreev D.E., Terenin I.M., Anisimova A.S., Shatsky I.N., Dmitriev S.E. (2019) A novel uORF-based regulatory mechanism controls translation of the human MDM2 and eIF2D mRNAs during stress. Biochimie. 157, 92–101.
  43. Dieudonné F.-X., O'Connor P.B.F., Gubler-Jaquier P., Yasrebi H., Conne B., Nikolaev S., Antonarakis S., Baranov P.V., Curran J. (2015) The effect of heterogeneous transcription start sites (TSS) on the translatome: implications for the mammalian cellular phenotype. BMC Genomics. 16, 986.
  44. Wang X., Hou J., Quedenau C., Chen W. (2016) Pervasive isoform‐specific translational regulation via alternative transcription start sites in mammals. Mol. Systems Biol. 12, 875
  45. Li H., Bai L., Li H., Li X., Kang Y., Zhang N., Sun J., Shao Z. (2019) Selective translational usage of TSS and core promoters revealed by translatome sequencing. BMC Genomics. 20, 282.
  46. Kozak M. (1991) A short leader sequence impairs the fidelity of initiation by eukaryotic ribosomes. Gene Expression. 1, 111–115.
  47. Elfakess R., Dikstein R. (2008) A translation initiation element specific to mRNAs with very short 5′UTR that also regulates transcription. PLoS One. 3, e3094.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Appendix Table S1
Download (296KB)
Indexing metadata
3. Fig. 1. The structure of eRF1 mRNA. a – Schematic diagram of the organization of the reference eRF1 mRNA NM_004730. The arrows indicate the primers used to obtain the 3'-UTR of eRF1 mRNA. b – Schematic diagram of the organization of the 5'-UTR of eRF1 and the frequency of transcription start site usage according to the DBTSS database. The start and stop codons are marked with green and red rectangles, respectively. The number inside the rectangles indicates the reading frame relative to the main ORF. The number next to the green squares indicates the AUG sequence number relative to the start codon of the main ORF, taken as 0. The sequence of the TISU regulatory element is highlighted in light green. The truncated sequence of the 5'-UTR of eRF1, studied in this work, is shown in gray, along with the full-length one.

Download (381KB)
Indexing metadata
4. Fig. 2. Effect of 5' UTR and eRF1 ORF, as well as excess eRF1, on read-through and translation. a – General scheme of constructs with premature stop codons upstream of nanoluciferase ORF. The construct includes 5' UTR and first 13 codons of β-globin mRNA ORF followed by stop/sense codon (UAA/AAA, UAG/UAU, UGA/UGU) and corresponding 6-nucleotide 3' context (GGGCUG, GAUAAU, CUAGUA) to enhance read-through, followed by Nluc ORF without its own start codon in the same reading frame. 3' UTR is artificial and is produced from ~170 nucleotides after the stop codon in Nluc ORF in the original plasmid. b – Schematic diagram of model constructs for studying read-through of eRF1 mRNA stop codons. c – Level of read-through of model mRNAs with a reporter containing nanoluciferase measured in cell-free translation systems. d – Effect of excess eRF1 on read-through of stop codons in models with 5'-UTR and ORF of native mRNA. d – Effect of eRF1 5'-UTR length on translation. The compared templates have identical sequences after the start of translation and differ only in the length of 5'-UTR. Relative luminescence units, n.a. – differences are not significant.

Download (360KB)
Indexing metadata
5. Fig. 3. Effect of 5′-UTR and 3′-UTR of eRF1 mRNA on translation. a – Schematic of constructs encoding firefly luciferase used in the experiments. b – Effect of 5′- and 3′-UTR of eRF1 mRNA on Fluc translation in HEK293 lysate. c – Effect of 5′- and 3′-UTR of eRF1 mRNA on Fluc translation in HEK293 cell culture.

Download (305KB)
Indexing metadata
6. Fig. 4. Effect of start and stop codons in the sORF 5′-UTR of eRF1 mRNA. a – General scheme of the constructs representing four mRNA variants: “WT” – wild type, eRF1 5′-UTR is unchanged and contains the start and stop codon of the sORF, “-start in 5′-UTR” – the start codon of the sORF is replaced by the codon AAG, “-stop in 5′-UTR” – the stop codon of the sORF is replaced by UCG, “-start and stop in 5′-UTR” – the start codon of the sORF is replaced by the codon AAG, and the stop codon is replaced by UCG. b – Efficiency of in vivo translation in HEK293 cells of reporter mRNA with mutations in the start and stop codon of the sORF 5′-UTR from eRF1 mRNA.

Download (158KB)
Indexing metadata

Copyright (c) 2024 Russian Academy of Sciences