Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

Autoři: Kotchaphorn Mangkalaphiban aff001;  Feng He aff001;  Robin Ganesan aff001;  Chan Wu aff001;  Richard Baker aff001;  Allan Jacobson aff001
Působiště autorů: Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America aff001
Vyšlo v časopise: Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae. PLoS Genet 17(4): e1009538. doi:10.1371/journal.pgen.1009538
Kategorie: Research Article
doi: 10.1371/journal.pgen.1009538


Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

Klíčová slova:

Gene expression – Genetic footprinting – Messenger RNA – Nucleotides – Ribosomes – Transfer RNA – Translation termination – Yeast


1. Hellen CUT. Translation Termination and Ribosome Recycling in Eukaryotes. Cold Spring Harb Perspect Biol. 2018 Oct;10(10):a032656. doi: 10.1101/cshperspect.a032656 29735640

2. Salas-Marco J, Bedwell DM. GTP Hydrolysis by eRF3 Facilitates Stop Codon Decoding during Eukaryotic Translation Termination. Mol Cell Biol. 2004 Sep 1;24(17):7769–78. doi: 10.1128/MCB.24.17.7769-7778.2004 15314182

3. Schuller AP, Green R. Roadblocks and resolutions in eukaryotic translation. Nat Rev Mol Cell Biol. 2018 Aug;19(8):526–41. doi: 10.1038/s41580-018-0011-4 29760421

4. Dabrowski M, Bukowy-Bieryllo Z, Zietkiewicz E. Translational readthrough potential of natural termination codons in eucaryotes–The impact of RNA sequence. RNA Biol. 2015 Sep 2;12(9):950–8. doi: 10.1080/15476286.2015.1068497 26176195

5. Rodnina MV, Korniy N, Klimova M, Karki P, Peng B-Z, Senyushkina T, et al. Translational recoding: canonical translation mechanisms reinterpreted. Nucleic Acids Res. 2020 Feb 20;48(3):1056–67. doi: 10.1093/nar/gkz783 31511883

6. Brenner S, Stretton AOW, Kaplan S. Genetic Code: The ‘Nonsense’ Triplets for Chain Termination and their Suppression. Nature. 1965 Jun;206(4988):994–8. doi: 10.1038/206994a0 5320272

7. Schueren F, Thoms S. Functional Translational Readthrough: A Systems Biology Perspective. Brosius J, editor. PLOS Genet. 2016 Aug 4;12(8):e1006196. doi: 10.1371/journal.pgen.1006196 27490485

8. Skuzeski JM, Nichols LM, Gesteland RF, Atkins JF. The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons. J Mol Biol. 1991 Mar 20;218(2):365–73. doi: 10.1016/0022-2836(91)90718-l 2010914

9. De Bellis M, Pisani F, Mola MG, Rosito S, Simone L, Buccoliero C, et al. Translational readthrough generates new astrocyte AQP4 isoforms that modulate supramolecular clustering, glial endfeet localization, and water transport: DE BELLIS et al. Glia. 2017 May;65(5):790–803. doi: 10.1002/glia.23126 28206694

10. Eswarappa SM, Potdar AA, Koch WJ, Fan Y, Vasu K, Lindner D, et al. Programmed Translational Readthrough Generates Antiangiogenic VEGF-Ax. Cell. 2014 Jun;157(7):1605–18. doi: 10.1016/j.cell.2014.04.033 24949972

11. Hofhuis J, Schueren F, Nötzel C, Lingner T, Gärtner J, Jahn O, et al. The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code. Open Biol. 2016 Nov;6(11):160246. doi: 10.1098/rsob.160246 27881739

12. Loughran G, Chou M-Y, Ivanov IP, Jungreis I, Kellis M, Kiran AM, et al. Evidence of efficient stop codon readthrough in four mammalian genes. Nucleic Acids Res. 2014 Aug 18;42(14):8928–38. doi: 10.1093/nar/gku608 25013167

13. Loughran G, Jungreis I, Tzani I, Power M, Dmitriev RI, Ivanov IP, et al. Stop codon readthrough generates a C-terminally extended variant of the human vitamin D receptor with reduced calcitriol response. J Biol Chem. 2018 Mar 23;293(12):4434–44. doi: 10.1074/jbc.M117.818526 29386352

14. Namy O, Duchateau-Nguyen G, Rousset J-P. Translational readthrough of the PDE2 stop codon modulates cAMP levels in Saccharomyces cerevisiae. Mol Microbiol. 2002;43(3):641–52. doi: 10.1046/j.1365-2958.2002.02770.x 11929521

15. Rajput B, Pruitt KD, Murphy TD. RefSeq curation and annotation of stop codon recoding in vertebrates. Nucleic Acids Res. 2019 Jan 25;47(2):594–606. doi: 10.1093/nar/gky1234 30535227

16. Schueren F, Lingner T, George R, Hofhuis J, Dickel C, Gärtner J, et al. Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals. eLife. 2014 Sep 23;3:e03640.

17. Steneberg P, Samakovlis C. A novel stop codon readthrough mechanism produces functional Headcase protein in Drosophila trachea. EMBO Rep. 2001 Jul 1;2(7):593–7. doi: 10.1093/embo-reports/kve128 11463742

18. Yamaguchi Y, Baba H. Phylogenetically Conserved Sequences Around Myelin P0 Stop Codon are Essential for Translational Readthrough to Produce L-MPZ. Neurochem Res. 2018 Jan;43(1):227–37. doi: 10.1007/s11064-017-2423-5 29081003

19. Dunn JG, Foo CK, Belletier NG, Gavis ER, Weissman JS. Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster. eLife. 2013 Dec 3;2:e01179. doi: 10.7554/eLife.01179 24302569

20. Jungreis I, Lin MF, Spokony R, Chan CS, Negre N, Victorsen A, et al. Evidence of abundant stop codon readthrough in Drosophila and other metazoa. Genome Res. 2011 Dec 1;21(12):2096–113. doi: 10.1101/gr.119974.110 21994247

21. Kleppe AS, Bornberg-Bauer E. Robustness by intrinsically disordered C-termini and translational readthrough. Nucleic Acids Res. 2018 Nov 2;46(19):10184–94. doi: 10.1093/nar/gky778 30247639

22. Tate WP, Cridge AG, Brown CM. ‘Stop’ in protein synthesis is modulated with exquisite subtlety by an extended RNA translation signal. Biochem Soc Trans. 2018 Nov 12;BST20180190. doi: 10.1042/BST20180190 30420414

23. Bonetti B, Fu L, Moon J, Bedwell DM. The Efficiency of Translation Termination is Determined by a Synergistic Interplay Between Upstream and Downstream Sequences inSaccharomyces cerevisiae. J Mol Biol. 1995 Aug;251(3):334–45. doi: 10.1006/jmbi.1995.0438 7650736

24. Mottagui-Tabar S, Tuite MF, Isaksson LA. The influence of 5’ codon context on translation termination in Saccharomyces cerevisiae. Eur J Biochem. 1998 Oct 1;257(1):249–54. doi: 10.1046/j.1432-1327.1998.2570249.x 9799126

25. Tork S, Hatin I, Rousset J-P, Fabret C. The major 5′ determinant in stop codon read-through involves two adjacent adenines. Nucleic Acids Res. 2004;32(2):415–21. doi: 10.1093/nar/gkh201 14736996

26. Anzalone AV, Zairis S, Lin AJ, Rabadan R, Cornish VW. Interrogation of Eukaryotic Stop Codon Readthrough Signals by in Vitro RNA Selection. Biochemistry. 2019 Feb 26;58(8):1167–78. doi: 10.1021/acs.biochem.8b01280 30698415

27. Cridge AG, Crowe-McAuliffe C, Mathew SF, Tate WP. Eukaryotic translational termination efficiency is influenced by the 3′ nucleotides within the ribosomal mRNA channel. Nucleic Acids Res. 2018 Feb 28;46(4):1927–44. doi: 10.1093/nar/gkx1315 29325104

28. Namy O, Hatin I, Rousset J-P. Impact of the six nucleotides downstream of the stop codon on translation termination. EMBO Rep. 2001 Sep 15;2(9):787–93. doi: 10.1093/embo-reports/kve176 11520858

29. Firth AE, Wills NM, Gesteland RF, Atkins JF. Stimulation of stop codon readthrough: frequent presence of an extended 3′ RNA structural element. Nucleic Acids Res. 2011 Aug;39(15):6679–91. doi: 10.1093/nar/gkr224 21525127

30. Ingolia NT, Ghaemmaghami S, Newman JRS, Weissman JS. Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling. Science. 2009 Apr 10;324(5924):218–23. doi: 10.1126/science.1168978 19213877

31. Li C, Zhang J. Stop-codon read-through arises largely from molecular errors and is generally nonadaptive. PLOS Genet. 2019 May 23;15(5):e1008141. doi: 10.1371/journal.pgen.1008141 31120886

32. Wangen JR, Green R. Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides. eLife. 2020 Jan 23;9:e52611. doi: 10.7554/eLife.52611 31971508

33. Brown A, Shao S, Murray J, Hegde RS, Ramakrishnan V. Structural basis for stop codon recognition in eukaryotes. Nature. 2015 Aug;524(7566):493–6. doi: 10.1038/nature14896 26245381

34. Shao S, Murray J, Brown A, Taunton J, Ramakrishnan V, Hegde RS. Decoding Mammalian Ribosome-mRNA States by Translational GTPase Complexes. Cell. 2016 Nov 17;167(5):1229–1240.e15. doi: 10.1016/j.cell.2016.10.046 27863242

35. Wilson DN, Arenz S, Beckmann R. Translation regulation via nascent polypeptide-mediated ribosome stalling. Curr Opin Struct Biol. 2016 Apr 1;37:123–33. doi: 10.1016/ 26859868

36. Ivanov A, Mikhailova T, Eliseev B, Yeramala L, Sokolova E, Susorov D, et al. PABP enhances release factor recruitment and stop codon recognition during translation termination. Nucleic Acids Res. 2016 Sep 19;44(16):7766–76. doi: 10.1093/nar/gkw635 27418677

37. Swart EC, Serra V, Petroni G, Nowacki M. Genetic Codes with No Dedicated Stop Codon: Context-Dependent Translation Termination. Cell. 2016 Jul 28;166(3):691–702. doi: 10.1016/j.cell.2016.06.020 27426948

38. Wu C, Roy B, He F, Yan K, Jacobson A. Poly(A)-Binding Protein Regulates the Efficiency of Translation Termination. Cell Rep. 2020 Nov;33(7):108399. doi: 10.1016/j.celrep.2020.108399 33207198

39. Stansfield I, Kushnirov VV, Jones KM, Tuite MF. A conditional-Lethal Translation Termination Defect in a sup45 Mutant of the Yeast Succhuromyces Cerevisiue. Eur J Biochem. 1997 May;245(3):557–63. doi: 10.1111/j.1432-1033.1997.00557.x 9182990

40. Wu CC-C, Zinshteyn B, Wehner KA, Green R. High-Resolution Ribosome Profiling Defines Discrete Ribosome Elongation States and Translational Regulation during Cellular Stress. Mol Cell. 2019 Mar;73(5):959–970.e5. doi: 10.1016/j.molcel.2018.12.009 30686592

41. Young DJ, Guydosh NR, Zhang F, Hinnebusch AG, Green R. Rli1/ABCE1 Recycles Terminating Ribosomes and Controls Translation Reinitiation in 3′UTRs In Vivo. Cell. 2015 Aug;162(4):872–84. doi: 10.1016/j.cell.2015.07.041 26276635

42. He F, Jacobson A. Nonsense-Mediated mRNA Decay: Degradation of Defective Transcripts Is Only Part of the Story. Annu Rev Genet. 2015 Nov 23;49(1):339–66. doi: 10.1146/annurev-genet-112414-054639 26436458

43. Celik A, Baker R, He F, Jacobson A. High-resolution profiling of NMD targets in yeast reveals translational fidelity as a basis for substrate selection. RNA. 2017 May;23(5):735–48. doi: 10.1261/rna.060541.116 28209632

44. Breiman L. Random Forests. Mach Learn. 2001 Oct 1;45(1):5–32.

45. Liaw A, Wiener M. Classification and Regression by randomForest. R News. 2002;2(3):18–22.

46. Lorenz R, Bernhart SH, Höner zu Siederdissen C, Tafer H, Flamm C, Stadler PF, et al. ViennaRNA Package 2.0. Algorithms Mol Biol. 2011 Nov 24;6(1):26. doi: 10.1186/1748-7188-6-26 22115189

47. Drozdetskiy A, Cole C, Procter J, Barton GJ. JPred4: a protein secondary structure prediction server. Nucleic Acids Res. 2015 Jul 1;43(W1):W389–94. doi: 10.1093/nar/gkv332 25883141

48. Pechmann S, Frydman J. Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding. Nat Struct Mol Biol. 2013 Feb;20(2):237–43. doi: 10.1038/nsmb.2466 23262490

49. Reis M dos, Savva R, Wernisch L. Solving the riddle of codon usage preferences: a test for translational selection. Nucleic Acids Res. 2004;32(17):5036–44. doi: 10.1093/nar/gkh834 15448185

50. Presnyak V, Alhusaini N, Chen Y-H, Martin S, Morris N, Kline N, et al. Codon Optimality Is a Major Determinant of mRNA Stability. Cell. 2015 Mar;160(6):1111–24. doi: 10.1016/j.cell.2015.02.029 25768907

51. Johansson MJO, Esberg A, Huang B, Björk GR, Byström AS. Eukaryotic Wobble Uridine Modifications Promote a Functionally Redundant Decoding System. Mol Cell Biol. 2008 May 15;28(10):3301–12. doi: 10.1128/MCB.01542-07 18332122

52. Beznosková P, Cuchalová L, Wagner S, Shoemaker CJ, Gunišová S, von der Haar T, et al. Translation Initiation Factors eIF3 and HCR1 Control Translation Termination and Stop Codon Read-Through in Yeast Cells. Hinnebusch AG, editor. PLoS Genet. 2013 Nov 21;9(11):e1003962. doi: 10.1371/journal.pgen.1003962 24278036

53. Khoshnevis S, Gross T, Rotte C, Baierlein C, Ficner R, Krebber H. The iron–sulphur protein RNase L inhibitor functions in translation termination. EMBO Rep. 2010 Mar;11(3):214–9. doi: 10.1038/embor.2009.272 20062004

54. Young DJ, Guydosh NR. Hcr1/eIF3j Is a 60S Ribosomal Subunit Recycling Accessory Factor In Vivo. Cell Rep. 2019 Jul 2;28(1):39–50.e4. doi: 10.1016/j.celrep.2019.05.111 31269449

55. Roque S, Cerciat M, Gaugué I, Mora L, Floch AG, Zamaroczy M de, et al. Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae. RNA. 2015 Jan 1;21(1):124–34. doi: 10.1261/rna.047282.114 25411355

56. Amrani N, Ganesan R, Kervestin S, Mangus DA, Ghosh S, Jacobson A. A faux 3′-UTR promotes aberrant termination and triggers nonsense- mediated mRNA decay. Nature. 2004 Nov;432(7013):112–8. doi: 10.1038/nature03060 15525991

57. He F, Li X, Spatrick P, Casillo R, Dong S, Jacobson A. Genome-Wide Analysis of mRNAs Regulated by the Nonsense-Mediated and 5′ to 3′ mRNA Decay Pathways in Yeast. Mol Cell. 2003 Dec;12(6):1439–52. doi: 10.1016/s1097-2765(03)00446-5 14690598

58. Ganesan R, Leszyk J, Jacobson A. Selective profiling of ribosomes associated with yeast Upf proteins. Methods. 2019 Feb 15;155:58–67. doi: 10.1016/j.ymeth.2018.12.008 30593864

59. Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc. 2012 Aug;7(8):1534–50. doi: 10.1038/nprot.2012.086 22836135

60. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal. 2011 May 2;17(1):10.

61. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009 Aug 15;25(16):2078–9. doi: 10.1093/bioinformatics/btp352 19505943

62. Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10(3):R25. doi: 10.1186/gb-2009-10-3-r25 19261174

63. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010 May;28(5):511–5. doi: 10.1038/nbt.1621 20436464

64. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011 Dec;12(1):323.

65. Nagalakshmi U, Wang Z, Waern K, Shou C, Raha D, Gerstein M, et al. The Transcriptional Landscape of the Yeast Genome Defined by RNA Sequencing. Science. 2008 Jun 6;320(5881):1344–9. doi: 10.1126/science.1158441 18451266

66. Lauria F, Tebaldi T, Bernabò P, Groen EJN, Gillingwater TH, Viero G. riboWaltz: Optimization of ribosome P-site positioning in ribosome profiling data. PLOS Comput Biol. 2018 Aug 13;14(8):e1006169. doi: 10.1371/journal.pcbi.1006169 30102689

67. Kuhn M. Building Predictive Models in R Using the caret Package. J Stat Softw. 2008;28(5).

68. James G, Witten D, Hastie T, Tibshirani R. An Introduction to Statistical Learning. New York, NY: Springer New York; 2013. (Springer Texts in Statistics; vol. 103).

69. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol. 2011 Jan;29(1):24–6. doi: 10.1038/nbt.1754 21221095

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