The molecular mechanism of stop codon recognition by the release factor eRF1 in complex with eRF3 has been described in great detail; however, our understanding of what determines the difference in termination efficiencies among various stop codon tetranucleotides and how near-cognate (nc) tRNAs recode stop codons during programmed readthrough in Saccharomyces cerevisiae is still poor. Here, we show that UGA-C as the only tetranucleotide of all four possible combinations dramatically exacerbated the readthrough phenotype of the stop codon recognition-deficient mutants in eRF1. Since the same is true also for UAA-C and UAG-C, we propose that the exceptionally high readthrough levels that all three stop codons display when followed by cytosine are partially caused by the compromised sampling ability of eRF1, which specifically senses cytosine at the +4 position. The difference in termination efficiencies among the remaining three UGA-N tetranucleotides is then given by their varying preferences for nc-tRNAs. In particular, UGA-A allows increased incorporation of Trp-tRNA whereas UGA-G and UGA-C favor Cys-tRNA. Our findings thus expand the repertoire of general decoding rules by showing that the +4 base determines the preferred selection of nc-tRNAs and, in the case of cytosine, it also genetically interacts with eRF1. Finally, using an example of the GCN4 translational control governed by four short uORFs, we also show how the evolution of this mechanism dealt with undesirable readthrough on those uORFs that serve as the key translation reinitiation promoting features of the GCN4 regulation, as both of these otherwise counteracting activities, readthrough versus reinitiation, are mediated by eIF3.

Laboratory of Regulation of Gene Expression Institute of Microbiology ASCR 142 20 Prague Czech Republic

Laboratory of Regulation of Gene Expression Institute of Microbiology ASCR 142 20 Prague Czech Republic Faculty of Science Charles University Prague 128 43 Prague Czech Republic

Zobrazit více v PubMed

Akhmaloka, Susilowati PE, Subandi, Madayanti F. 2008. Mutation at tyrosine in AMLRY (GILRY like) motif of yeast eRF1 on nonsense codons suppression and binding affinity to eRF3. Int J Biol Sci 4: 87–95. PubMed PMC

Bertram G, Bell HA, Ritchie DW, Fullerton G, Stansfield I. 2000. Terminating eukaryote translation: domain 1 of release factor eRF1 functions in stop codon recognition. RNA 6: 1236–1247. PubMed PMC

Beznosková P, Cuchalová L, Wagner S, Shoemaker CJ, Gunišová S, Von der Haar T, Valášek LS. 2013. Translation initiation factors eIF3 and HCR1 control translation termination and stop codon read-through in yeast cells. PLoS Genet 9: e1003962. PubMed PMC

Beznosková P, Wagner S, Jansen ME, von der Haar T, Valášek LS. 2015. Translation initiation factor eIF3 promotes programmed stop codon readthrough. Nucleic Acids Res 43: 5099–5111. PubMed PMC

Bidou L, Allamand V, Rousset JP, Namy O. 2012. Sense from nonsense: therapies for premature stop codon diseases. Trends Mol Med 18: 679–688. PubMed

Blanchet S, Cornu D, Argentini M, Namy O. 2014. New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae. Nucleic Acids Res 42: 10061–10072. PubMed PMC

Bonetti B, Fu L, Moon J, Bedwell DM. 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. PubMed

Brown A, Shao S, Murray J, Hegde RS, Ramakrishnan V. 2015. Structural basis for stop codon recognition in eukaryotes. Nature 524: 493–496. PubMed PMC

Bulygin KN, Repkova MN, Ven'yaminova AG, Graifer DM, Karpova GG, Frolova LY, Kisselev LL. 2002. Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in 80S ribosomes. FEBS Lett 514: 96–101. PubMed

Cross FR, Tinkelenberg AH. 1991. A potential positive feedback loop controlling CLN1 and CLN2 gene expression at the start of the yeast cell cycle. Cell 65: 875–883. PubMed

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. PubMed PMC

des Georges A, Hashem Y, Unbehaun A, Grassucci RA, Taylor D, Hellen CU, Pestova TV, Frank J. 2014. Structure of the mammalian ribosomal pre-termination complex associated with eRF1•eRF3•GDPNP. Nucleic Acids Res 42: 3409–3418. PubMed PMC

Dinman JD. 2012. Control of gene expression by translational recoding. Adv Protein Chem Struct Biol 86: 129–149. PubMed PMC

Erzberger JP, Stengel F, Pellarin R, Zhang S, Schaefer T, Aylett CH, Cimermancic P, Boehringer D, Sali A, Aebersold R, et al. 2014. Molecular architecture of the 40S•eIF1•eIF3 translation initiation complex. Cell 158: 1123–1135. PubMed PMC

Grant CM, Hinnebusch AG. 1994. Effect of sequence context at stop codons on efficiency of reinitiation in GCN4 translational control. Mol Cell Biol 14: 606–618. PubMed PMC

Gunišová S, Valášek LS. 2014. Fail-safe mechanism of GCN4 translational control—uORF2 promotes reinitiation by analogous mechanism to uORF1 and thus secures its key role in GCN4 expression. Nucleic Acids Res 42: 5880–5893. PubMed PMC

Hinnebusch AG. 2005. Translational regulation of GCN4 and the general amino acid control of yeast. Annu Rev Microbiol 59: 407–450. PubMed

Jackson RJ, Hellen CU, Pestova TV. 2012. Termination and post-termination events in eukaryotic translation. Adv Protein Chem Struct Biol 86: 45–93. PubMed

Keeling KM, Lanier J, Du M, Salas-Marco J, Gao L, Kaenjak-Angeletti A, Bedwell DM. 2004. Leaky termination at premature stop codons antagonizes nonsense-mediated mRNA decay in S. cerevisiae. RNA 10: 691–703. PubMed PMC

Keeling KM, Xue X, Gunn G, Bedwell DM. 2014. Therapeutics based on stop codon readthrough. Annu Rev Genomics Hum Genet 15: 371–394. PubMed PMC

Lee HL, Dougherty JP. 2012. Pharmaceutical therapies to recode nonsense mutations in inherited diseases. Pharmacol Ther 136: 227–266. PubMed

Linde L, Kerem B. 2008. Introducing sense into nonsense in treatments of human genetic diseases. Trends Genet 24: 552–563. PubMed

Matheisl S, Berninghausen O, Becker T, Beckmann R. 2015. Structure of a human translation termination complex. Nucleic Acids Res 43: 8615–8626. PubMed PMC

McCaughan KK, Brown CM, Dalphin ME, Berry MJ, Tate WP. 1995. Translational termination efficiency in mammals is influenced by the base following the stop codon. Proc Natl Acad Sci 92: 5431–5435. PubMed PMC

Merritt GH, Naemi WR, Mugnier P, Webb HM, Tuite MF, von der Haar T. 2010. Decoding accuracy in eRF1 mutants and its correlation with pleiotropic quantitative traits in yeast. Nucleic Acids Res 38: 5479–5492. PubMed PMC

Mort M, Ivanov D, Cooper DN, Chuzhanova NA. 2008. A meta-analysis of nonsense mutations causing human genetic disease. Hum Mutat 29: 1037–1047. PubMed

Mottagui-Tabar S, Tuite MF, Isaksson LA. 1998. The influence of 5′ codon context on translation termination in Saccharomyces cerevisiae. Eur J Biochem 257: 249–254. PubMed

Muhlrad D, Parker R. 1999. Recognition of yeast mRNAs as “nonsense containing” leads to both inhibition of mRNA translation and mRNA degradation: implications for the control of mRNA decapping. Mol Biol Cell 10: 3971–3978. PubMed PMC

Munzarová V, Pánek J, Gunišová S, Dányi I, Szamecz B, Valášek LS. 2011. Translation reinitiation relies on the interaction between eIF3a/TIF32 and progressively folded cis-acting mRNA elements preceding short uORFs. PLoS Genet 7: e1002137. PubMed PMC

Namy O, Hatin I, Rousset JP. 2001. Impact of the six nucleotides downstream of the stop codon on translation termination. EMBO Rep 2: 787–793. PubMed PMC

Pechmann S, Frydman J. 2013. Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding. Nat Struct Mol Biol 20: 237–243. PubMed PMC

Phillips-Jones MK, Hill LS, Atkinson J, Martin R. 1995. Context effects on misreading and suppression at UAG codons in human cells. Mol Cell Biol 15: 6593–6600. PubMed PMC

Pineyro D, Torres AG, de Pouplana LR. 2014. Biogenesis and evolution of functional tRNAs. In Fungal RNA biology (ed. Sesma A, von der Haar T), pp. 233–267. Springer International Publishing, Switzerland.

Pöyry TA, Kaminski A, Jackson RJ. 2004. What determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame? Genes Dev 18: 62–75. PubMed PMC

Roy B, Leszyk JD, Mangus DA, Jacobson A. 2015. Nonsense suppression by near-cognate tRNAs employs alternative base pairing at codon positions 1 and 3. Proc Natl Acad Sci 112: 3038–3043. PubMed PMC

Szamecz B, Rutkai E, Cuchalová L, Munzarová V, Herrmannová A, Nielsen KH, Burela L, Hinnebusch AG, Valášek L. 2008. eIF3a cooperates with sequences 5′ of uORF1 to promote resumption of scanning by post-termination ribosomes for reinitiation on GCN4 mRNA. Genes Dev 22: 2414–2425. PubMed PMC

Valášek LS. 2012. ‘Ribozoomin’—translation initiation from the perspective of the ribosome-bound eukaryotic initiation factors (eIFs). Curr Protein Pept Sci 13: 305–330. PubMed PMC

Valášek L, Mathew A, Shin BS, Nielsen KH, Szamecz B, Hinnebusch AG. 2003. The yeast eIF3 subunits TIF32/a, NIP1/c, and eIF5 make critical connections with the 40S ribosome in vivo. Genes Dev 17: 786–799. PubMed PMC

von der Haar T, Tuite MF. 2007. Regulated translational bypass of stop codons in yeast. Trends Microbiol 15: 78–86. PubMed

von der Haar T, Valasek LS. 2014. mRNA translation: fungal variations on a eukaryotic theme. Springer International Publishing, Switzerland.

Zaher HS, Green R. 2009. Fidelity at the molecular level: lessons from protein synthesis. Cell 136: 746–762. PubMed PMC

Ribosomal A-site interactions with near-cognate tRNAs drive stop codon readthrough

Differential effects of 40S ribosome recycling factors on reinitiation at regulatory uORFs in GCN4 mRNA are not dictated by their roles in bulk 40S recycling

Impacts of yeast Tma20/MCTS1, Tma22/DENR and Tma64/eIF2D on translation reinitiation and ribosome recycling

Cysteine tRNA acts as a stop codon readthrough-inducing tRNA in the human HEK293T cell line

Short tRNA anticodon stem and mutant eRF1 allow stop codon reassignment

Increased expression of tryptophan and tyrosine tRNAs elevates stop codon readthrough of reporter systems in human cell lines

uS3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3

Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes

Yeast applied readthrough inducing system (YARIS): an invivo assay for the comprehensive study of translational readthrough

Please do not recycle! Translation reinitiation in microbes and higher eukaryotes

Embraced by eIF3: structural and functional insights into the roles of eIF3 across the translation cycle

Human eIF3b and eIF3a serve as the nucleation core for the assembly of eIF3 into two interconnected modules: the yeast-like core and the octamer

In-depth analysis of cis-determinants that either promote or inhibit reinitiation on GCN4 mRNA after translation of its four short uORFs