Recognition of 3′ nucleotide context and stop codon readthrough are determined during mRNA translation elongation
2022; Elsevier BV; Volume: 298; Issue: 7 Linguagem: Inglês
10.1016/j.jbc.2022.102133
ISSN1083-351X
AutoresNikita Biziaev, Elizaveta Sokolova, Dmitry V. Yanvarev, I. Yu. Toropygin, Alexey Shuvalov, Tatiana Egorova, Elena Alkalaeva,
Tópico(s)RNA Research and Splicing
ResumoThe nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3′ contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3′ stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3′ nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3′ nucleotides. Moreover, the efficiency of translation termination in weak 3′ contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3′ nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs. The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3′ contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3′ stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3′ nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3′ nucleotides. Moreover, the efficiency of translation termination in weak 3′ contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3′ nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs. Protein synthesis is completed when the stop codon (UAA, UAG, or UGA) occupies the ribosomal A site, where the eukaryotic release factors (eRFs) decode it. In eukaryotes, the tRNA-mimicking factor eRF1 recognizes all three stop codons and promotes the release of the synthesized peptide from the peptidyl-transferase center. It is stimulated by eRF3, which resembles the elongation factor 1A (eEF1A) (1Zhouravleva G. Frolova L. Le Goff X. Le Guellec R. Inge-Vechtomov S. Kisselev L. et al.Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3.EMBO J. 1995; 14: 4065-4072Crossref PubMed Scopus (511) Google Scholar, 2Stansfield I. Jones K.M. Ter-Avanesyan M.D. Tuite5 M.F. The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae.EMBO J. 1995; 14: 4365-4373Crossref PubMed Scopus (422) Google Scholar, 3Alkalaeva E.Z. Pisarev A.V. Frolova L.Y. Kisselev L.L. Pestova T.V. In Vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3.Cell. 2006; 125: 1125-1136Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 4Nakamura Y. Ito K. Ehrenberg M. Mimicry grasps reality in translation termination.Cell. 2000; 101: 349-352Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Remarkably, positional and conformational similarities exist between the termination eRF1–eRF3 complex and elongation aa tRNA–eEF1 complexes (5Voorhees R.M. Schmeing T.M. Kelley A.C. Ramakrishnan V. The mechanism for activation of GTP hydrolysis on the ribosome.Science. 2010; 330: 835-838Crossref PubMed Scopus (273) Google Scholar, 6des Georges A. Hashem Y. Unbehaun A. Grassucci R.A. Taylor D. Hellen C.U.T. et al.Structure of the mammalian ribosomal pre-termination complex associated with eRF1•eRF3•GDPNP.Nucl. Acids Res. 2014; 42: 3409-3418Crossref PubMed Scopus (54) Google Scholar). Stop codon recognition is implemented by the conserved TASNIKS and YxCxxxF motifs of the N-domain of eRF1 (7Bertram G. Bell H.A. Ritchie D.W. Fullerton G. Stansfield I. Terminating eukaryote translation: domain 1 of release factor eRF1 functions in stop codon recognition.RNA. 2000; 6: 1236-1247Crossref PubMed Scopus (150) Google Scholar, 8Ito K. Frolova L. Seit-Nebi A. Karamyshev A. Kisselev L. Nakamura Y. Omnipotent decoding potential resides in eukaryotic translation termination factor eRF1 of variant-code organisms and is modulated by the interactions of amino acid sequences within domain 1.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8494-8499Crossref PubMed Scopus (73) Google Scholar). The lysine of the NIKS motif can be hydroxylated, which improves termination efficiency (9Feng T. Yamamoto A. Wilkins S.E. Sokolova E. Yates L.A. Münzel M. et al.Optimal translational termination requires C4 lysyl hydroxylation of eRF1.Mol. Cell. 2014; 53: 645-654Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). cryo-EM structures of mammalian ribosomal complexes containing a stop codon at the A site have shown that binding of eRF1 leads to changes in mRNA configuration so that the fourth nucleotide following the three bases of the stop codon is pulled into the A site (10Brown A. Shao S. Murray J. Hegde R.S. Ramakrishnan V. Structural basis for stop codon recognition in eukaryotes.Nature. 2015; 524: 493-496Crossref PubMed Scopus (164) Google Scholar, 11Matheisl S. Berninghausen O. Becker T. Beckmann R. Structure of a human translation termination complex.Nucl. Acids Res. 2015; 43: 8615-8626Crossref PubMed Scopus (73) Google Scholar, 12Shao S. Murray J. Brown A. Taunton J. Ramakrishnan V. Hegde R.S. Decoding mammalian ribosome-mRNA states by translational GTPase complexes.Cell. 2016; 167: 1229-1240.e15Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). This configuration differs from the shape of sense codons recognized by tRNAs and implicates a complex three-dimensional interplay of eRF1 and 18S rRNA. This also explains the strong impact of the identity of the 3′ nucleotide downstream of the stop codon on termination (13McCaughan K.K. Brown C.M. Dalphin M.E. Berry M.J. Tate W.P. Translational termination efficiency in mammals is influenced by the base following the stop codon.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5431-5435Crossref PubMed Scopus (222) Google Scholar). However, in some instances, the amino acid is incorporated into the nascent polypeptide chain instead of proper translation termination. Such an event is a result of stop codon suppression or readthrough, when the stop codon in the ribosomal A site is interpreted as a sense codon and is recognized by near-cognate tRNAs instead of eRF1. The basal level of stop codon readthrough commonly has a frequency of < 0.1% (14Keeling K.M. Xue X. Gunn G. Bedwell D.M. Therapeutics based on stop codon readthrough.Annu. Rev. Genomics Hum. Genet. 2014; 15: 371-394Crossref PubMed Scopus (168) Google Scholar), although in some cases, the level of readthrough was shown to be higher than 10% (15Loughran G. Chou M.Y. Ivanov I.P. Jungreis I. Kellis M. Kiran A.M. et al.Evidence of efficient stop codon readthrough in four mammalian genes.Nucl. Acids Res. 2014; 42: 8928-8938Crossref PubMed Scopus (123) Google Scholar, 16Schueren 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; 3e03640Crossref PubMed Scopus (106) Google Scholar). Phylogenetic analysis of the 12 Drosophila species revealed more than 280 conserved stop codon readthroughs. This was confirmed by ribosome profiling analysis, which indicated that readthrough was a relatively common event (17Jungreis I. Lin M.F. Spokony R. Chan C.S. Negre N. Victorsen A. et al.Evidence of abundant stop codon readthrough in Drosophila and other metazoa.Genome Res. 2011; 21: 2096-2113Crossref PubMed Scopus (135) Google Scholar, 18Dunn J.G. Foo C.K. Belletier N.G. Gavis E.R. Weissman J.S. Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster.Elife. 2013; 2e01179Crossref PubMed Scopus (232) Google Scholar). Subsequent studies have identified readthrough in fungi (19Freitag J. Ast J. Bölker M. Cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi.Nature. 2012; 485: 522-525Crossref PubMed Scopus (115) Google Scholar), and numerous studies have described readthrough for a large number of transcripts in mammals (15Loughran G. Chou M.Y. Ivanov I.P. Jungreis I. Kellis M. Kiran A.M. et al.Evidence of efficient stop codon readthrough in four mammalian genes.Nucl. Acids Res. 2014; 42: 8928-8938Crossref PubMed Scopus (123) Google Scholar, 16Schueren 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; 3e03640Crossref PubMed Scopus (106) Google Scholar, 18Dunn J.G. Foo C.K. Belletier N.G. Gavis E.R. Weissman J.S. Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster.Elife. 2013; 2e01179Crossref PubMed Scopus (232) Google Scholar, 20Chittum H.S. Lane W.S. Carlson B.A. Roller P.P. Lung F.D.T. Lee B.J. et al.Rabbit β-globin is extended beyond its UGA stop codon by multiple suppressions and translational reading gaps.Biochemistry. 1998; 37: 10866-10870Crossref PubMed Scopus (184) Google Scholar, 21Yamaguchi Y. Hayashi A. Campagnoni C.W. Kimura A. Inuzuka T. Baba H. L-MPZ, a novel isoform of myelin P0, is produced by stop codon readthrough.J. Biol. Chem. 2012; 287: 17765-17776Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 22Eswarappa S.M. Potdar A.A. Koch W.J. Fan Y. Vasu K. Lindner D. et al.Programmed translational readthrough generates antiangiogenic VEGF-Ax.Cell. 2014; 157: 1605-1618Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 23Loughran G. Jungreis I. Tzani I. Power M. Dmitriev R.I. Ivanov I.P. 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; 293: 4434-4444Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). In addition, premature stop codons also sustain a sufficient percentage of readthrough (<1%) (14Keeling K.M. Xue X. Gunn G. Bedwell D.M. Therapeutics based on stop codon readthrough.Annu. Rev. Genomics Hum. Genet. 2014; 15: 371-394Crossref PubMed Scopus (168) Google Scholar), which is essential for disease therapeutics, occurring as a result of nonsense mutations. These data demonstrate the importance of readthrough events, which can be considered not only as an error during the termination process but also as an important regulatory mechanism. It has been shown that the nucleotide context of stop codons significantly affects the readthrough level in different groups of eukaryotes (24Brar G.A. Beyond the triplet code: context cues transform translation.Cell. 2016; 167: 1681-1692Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 25Dabrowski M. Bukowy-Bieryllo Z. Zietkiewicz E. Translational readthrough potential of natural termination codons in eucaryotes – the impact of RNA sequence.RNA Biol. 2015; 12: 950-958Crossref PubMed Scopus (85) Google Scholar, 26Baranov P.V. Atkins J.F. Yordanova M.M. Augmented genetic decoding: global, local and temporal alterations of decoding processes and codon meaning.Nat. Rev. Genet. 2015; 16: 517-529Crossref PubMed Scopus (47) Google Scholar). The nearest 5′ and 3′ nucleotides of stop codons can decrease and increase the translation termination efficiency (27Bertram G. Innes S. Minella O. Richardson J. Stansfield I. Endless possibilities: translation termination and stop codon recognition.Microbiology. 2001; 147: 255-269Crossref PubMed Scopus (110) Google Scholar). Previously, the strongest influence on translation termination was demonstrated for +4 nucleotides immediately following the stop codon (13McCaughan K.K. Brown C.M. Dalphin M.E. Berry M.J. Tate W.P. Translational termination efficiency in mammals is influenced by the base following the stop codon.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5431-5435Crossref PubMed Scopus (222) Google Scholar, 28Pedersen W.T. Curran J.F. Effects of the nucleotide 3??? To an amber codon on ribosomal selection rates of suppressor tRNA and release factor-1.J. Mol. Biol. 1991; 219: 231-241Crossref PubMed Scopus (65) Google Scholar, 29Skuzeski J.M. Nichols L.M. Gesteland R.F. Atkins J.F. The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons.J. Mol. Biol. 1991; 218: 365-373Crossref PubMed Scopus (179) Google Scholar, 30Li G. Rice C.M. The signal for translational readthrough of a UGA codon in Sindbis virus RNA involves a single cytidine residue immediately downstream of the termination codon.J. Virol. 1993; 67: 5062-5067Crossref PubMed Scopus (80) Google Scholar, 31Poole E.S. Major L.L. Mannering S.A. Tate W.P. Translational termination in Escherichia coli: three bases following the stop codon crosslink to release factor 2 and affect the decoding efficiency of UGA-containing signals.Nucl. Acids Res. 1998; 26: 954-960Crossref PubMed Scopus (61) Google Scholar, 32Namy O. Hatin I. Rousset J.P. Impact of the six nucleotides downstream of the stop codon on translation termination.EMBO Rep. 2001; 2: 787-793Crossref PubMed Scopus (150) Google Scholar, 33Cavener D.R. Ray S.C. Eukaryotic start and stop translation sites.Nucl. Acids Res. 1991; 19: 3185-3192Crossref PubMed Scopus (526) Google Scholar). Few studies investigating the influence of the 3′ context have suggested that terminating signals include six nucleotides (31Poole E.S. Major L.L. Mannering S.A. Tate W.P. Translational termination in Escherichia coli: three bases following the stop codon crosslink to release factor 2 and affect the decoding efficiency of UGA-containing signals.Nucl. Acids Res. 1998; 26: 954-960Crossref PubMed Scopus (61) Google Scholar, 32Namy O. Hatin I. Rousset J.P. Impact of the six nucleotides downstream of the stop codon on translation termination.EMBO Rep. 2001; 2: 787-793Crossref PubMed Scopus (150) Google Scholar). Purines are preferred over pyrimidines in eukaryotic genomes (33Cavener D.R. Ray S.C. Eukaryotic start and stop translation sites.Nucl. Acids Res. 1991; 19: 3185-3192Crossref PubMed Scopus (526) Google Scholar, 34Cridge A.G. Major L.L. Mahagaonkar A.A. Poole E.S. Isaksson L.A. Tate W.P. Comparison of characteristics and function of translation termination signals between and within prokaryotic and eukaryotic organisms.Nucl. Acids Res. 2006; 34: 1959-1973Crossref PubMed Scopus (41) Google Scholar). Additionally, nucleotide distribution up to +9 in Saccharomyces cerevisiae and most likely in all eukaryotes is not random. It was demonstrated that positions +4, +5, +6, +8, and +9 were the key, and the +7 position did not have any effect (35Williams I. Richardson J. Starkey A. Stansfield I. Genome-wide prediction of stop codon readthrough during translation in the yeast Saccharomyces cerevisiae.Nucl. Acids Res. 2004; 32: 6605-6616Crossref PubMed Scopus (53) Google Scholar). The most effective suppression motif, CAA UUA entirely conforms to the 3′ context of stop codon UAG of tobacco mosaic virus (TMV). In mammalian cells, Cridge et al. (36Cridge A.G. Crowe-McAuliffe C. Mathew S.F. Tate W.P. Eukaryotic translational termination efficiency is influenced by the 3′ nucleotides within the ribosomal mRNA channel.Nucl. Acids Res. 2018; 46: 1927-1944Crossref PubMed Scopus (46) Google Scholar) affirmed the high impact on the readthrough of +4 and +8 nucleotides independently of the type of stop codon, and +5 and +6 positions determined the increase or decrease in readthrough depending on the stop codon and +4 nucleotides. There is evidence that different factors can influence readthrough levels in cooperation with the stop codon context. It has been shown that the eukaryotic translation initiation factor eIF3 increases readthrough in weak termination contexts, possibly promoting the incorporation of near-cognate tRNAs (37Beznosková P. Wagner S. Jansen M.E. Von Der Haar T. Valášek L.S. Translation initiation factor eIF3 promotes programmed stop codon readthrough.Nucl. Acids Res. 2015; 43: 5099-5111Crossref PubMed Scopus (62) Google Scholar). The posttranslational hydroxylation of prolyl in ribosomal protein Rps23 of the 40S subunit can also modulate termination accuracy in a context-dependent manner (38Loenarz C. Sekirnik R. Thalhammer A. Ge W. Spivakovsky E. Mackeen M.M. et al.Hydroxylation of the eukaryotic ribosomal decoding center affects translational accuracy.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 4019-4024Crossref PubMed Scopus (87) Google Scholar). However, the exact mechanism underlying the effect of the 3′ stop codon context on translation termination remains unknown. According to our previous study (39Sokolova E.E. Vlasov P.K. Egorova T.V. Shuvalov A.V. Alkalaeva E.Z. The influence of A/G composition of 3' stop codon contexts on translation termination efficiency in eukaryotes.Mol. Biol. 2020; 54: 739-748Crossref Scopus (1) Google Scholar), there is no apparent connection between nucleotide frequencies in the 3′ stop codon context and their effect on peptide-release efficiency. We investigated the effects of several 3′ stop codon contexts on readthrough and peptide release in the reconstituted translation termination system and revealed the molecular mechanism of this process. To study the mechanism underlying stop codon readthrough, we constructed model mRNAs containing two stop codons separated by hexanucleotide sequences (Fig. 1A). After the first stop codon (UAA, UAG, or UGA), we inserted several hexanucleotide sequences reported to be preferable for readthrough (15Loughran G. Chou M.Y. Ivanov I.P. Jungreis I. Kellis M. Kiran A.M. et al.Evidence of efficient stop codon readthrough in four mammalian genes.Nucl. Acids Res. 2014; 42: 8928-8938Crossref PubMed Scopus (123) Google Scholar, 29Skuzeski J.M. Nichols L.M. Gesteland R.F. Atkins J.F. The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons.J. Mol. Biol. 1991; 218: 365-373Crossref PubMed Scopus (179) Google Scholar, 40Bidou L. Hatin I. Perez N. Allamand V. Panthier J. Rousset J. Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment.Gene Ther. 2004; 11: 619-627Crossref PubMed Scopus (106) Google Scholar). We chose the most frequent stop codon, UAA, to be the second stop codon to exclude secondary readthrough and estimate the level of the first stop codon readthrough. The 3′ context of the second stop codon was A-rich AAG CUU, which ensured efficient translation termination according to our data (39Sokolova E.E. Vlasov P.K. Egorova T.V. Shuvalov A.V. Alkalaeva E.Z. The influence of A/G composition of 3' stop codon contexts on translation termination efficiency in eukaryotes.Mol. Biol. 2020; 54: 739-748Crossref Scopus (1) Google Scholar). After termination at the first stop codon, we obtained the MVHL tetrapeptide, and after readthrough, we obtained the heptapeptide MVHLXXX (Fig. 1A). To estimate the stop codon readthrough efficiency, we performed a fluorescent toe-printing assay of the ribosomal complexes assembled at the model mRNA. Fluorescent toe-printing is based on a reverse transcription reaction with fluorescently labeled primers that anneal downstream of the ribosomal complex (Fig. 1B). The position of the bound ribosome on the mRNA was then determined by the length of the fragment which extended the primer during reverse transcription. The consensus sequence CUAG, following the UGA stop codon, was previously shown to stimulate readthrough in a few mammalian genes (15Loughran G. Chou M.Y. Ivanov I.P. Jungreis I. Kellis M. Kiran A.M. et al.Evidence of efficient stop codon readthrough in four mammalian genes.Nucl. Acids Res. 2014; 42: 8928-8938Crossref PubMed Scopus (123) Google Scholar). Based on this sequence, we designed a model mRNA with a CUA GUA (Weak1) context located between the UGA and UAA stop codons. We reconstituted translation initiation 48S and 80S complexes on the mRNA (Fig. 1C) in the presence of the initiator methionyl-tRNA using an in vitro translation system (3Alkalaeva E.Z. Pisarev A.V. Frolova L.Y. Kisselev L.L. Pestova T.V. In Vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3.Cell. 2006; 125: 1125-1136Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). We then added Val-tRNAVal and translation elongation factors eEF1 and eEF2 in the presence of GTP to the initiation complex (IC). We obtained an elongation complex with dipeptidyl-tRNA (MV-tRNA) located at the P site. The addition of His-tRNAHis moved the ribosome to the next codon, and we obtained MVH-tRNA at the P site. The final addition of Leu-tRNALeu caused the formation of pretermination complex 1 (preTC1) with MVHL-tRNA at the P-site and UGA stop codon at the A site. Total rabbit aminoacylated (a.a.) tRNA moved preTC1 through the first stop codon to the second stop codon, giving the appearance of a +9 nucleotide peak (Fig. 1C), indicating the formation of preTC2. Intermediate peaks corresponding to elongation complexes that stopped at the CUA and GUA codons were also detected. Therefore, in the absence of eRFs, stop codon could be recognized by suppressor or near-cognate tRNAs at least in the specific 3′ nucleotide context. We suggest that the stop codon readthrough efficiency is determined by the presence of such tRNAs in the cell. In this experiment rabbit tRNAs and rabbit ribosomes were used (Fig. 1C). To determine the effect of tRNAs from different organisms on readthrough, we compared the effects of calf, rabbit, and yeast total tRNA (Fig. S1). It appeared that all tested total tRNA induced UGA readthrough. This indicates that they contain a sufficient amount of suppressor or near-cognate tRNA to recognize the UGA stop codon. However, the calf tRNA was less active than the others which means that it contained smaller amounts of appropriate tRNA. It is noteworthy that the second stop codon UAA in the strong A-rich 3′ context (39Sokolova E.E. Vlasov P.K. Egorova T.V. Shuvalov A.V. Alkalaeva E.Z. The influence of A/G composition of 3' stop codon contexts on translation termination efficiency in eukaryotes.Mol. Biol. 2020; 54: 739-748Crossref Scopus (1) Google Scholar) was not recognized by any of the tested preparations of tRNA. Therefore, different stop codons have different readthrough potentials. To determine the factors affecting the efficiency of stop codon readthrough in the absence of eRFs, we compared how all three stop codons in the Standard and Weak1 3′ contexts were decoded (Fig. 2A, row data are presented in Fig. S2). The 3ʹ context UGU GUG was chosen as the Standard. This context has been used in all our previous studies in the reconstituted mammalian translation system (3Alkalaeva E.Z. Pisarev A.V. Frolova L.Y. Kisselev L.L. Pestova T.V. In Vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3.Cell. 2006; 125: 1125-1136Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 39Sokolova E.E. Vlasov P.K. Egorova T.V. Shuvalov A.V. Alkalaeva E.Z. The influence of A/G composition of 3' stop codon contexts on translation termination efficiency in eukaryotes.Mol. Biol. 2020; 54: 739-748Crossref Scopus (1) Google Scholar, 41Alkalaeva E. Eliseev B. Ambrogelly A. Vlasov P. Kondrashov F.A. Gundllapalli S. et al.Translation termination in pyrrolysine-utilizing archaea.FEBS Lett. 2009; 583: 3455-3460Crossref PubMed Scopus (19) Google Scholar, 42Ivanov 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.Nucl. Acids Res. 2016; 44: 7766-7776Crossref PubMed Scopus (61) Google Scholar, 43Kryuchkova P. Grishin A. Eliseev B. Karyagina A. Frolova L. Alkalaeva E. Two-step model of stop codon recognition by eukaryotic release factor eRF1.Nucl. Acids Res. 2013; 41: 4573-4586Crossref PubMed Scopus (39) Google Scholar, 44Susorov D. Mikhailova T. Ivanov A. Sokolova E. Alkalaeva E. Stabilization of eukaryotic ribosomal termination complexes by deacylated tRNA.Nucl. Acids Res. 2015; 43: 3332-3343Crossref PubMed Scopus (12) Google Scholar, 45Egorova T. Biziaev N. Shuvalov A. Sokolova E. Mukba S. Evmenov K. et al.eIF3j facilitates loading of release factors into the ribosome.Nucl. Acids Res. 2021; 49: 11181-11196Crossref PubMed Scopus (3) Google Scholar, 46Shuvalova E. Egorova T. Ivanov A. Shuvalov A. Biziaev N. Mukba S. et al.Discovery of a novel role of tumor suppressor PDCD4 in stimulation of translation termination.J. Biol. Chem. 2021; 297101269Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, 47Ivanov A. Shuvalova E. Egorova T. Shuvalov A. Sokolova E. Bizyaev N. et al.Polyadenylate-binding protein-interacting proteins PAIP1 and PAIP2 affect translation termination.J. Biol. Chem. 2019; 294: 8630-8639Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The frequencies of triplets from this sequence in the human genome calculated earlier (1–1.8%) were close to that for random triplet NNN 1/64 = 1.56% (39Sokolova E.E. Vlasov P.K. Egorova T.V. Shuvalov A.V. Alkalaeva E.Z. The influence of A/G composition of 3' stop codon contexts on translation termination efficiency in eukaryotes.Mol. Biol. 2020; 54: 739-748Crossref Scopus (1) Google Scholar). Therefore, this sequence can be used as a control sequence. To exclude mistakes in all cases where we observed the appearance of intermediate peaks between the first and second stop codons, which indicated a lack of complementary tRNA, we determined the efficiency of readthrough by summarizing all the peaks appearing after the first stop codon. Thus, the effect of deficiency of the corresponding tRNAs in the calculations was reduced. For stop codons in the Standard context, readthrough varied from 0.5% to 28% (Table 1 and Fig. 2A). We observed that UAA was the strongest stop codon and UGA was the weakest. We determine the weakness of the stop codon based on the readthrough frequency. In the Standard context, the readthrough efficiency of these codons differed by a factor of 55 (Table 1). Stop codons in the Weak1 context demonstrated the same readthrough dependence: UAA was the strongest and UGA was the weakest stop codon. However, the overall readthrough rate was higher, ranging from 5% for UAA to 57% for UGA. Therefore, the readthrough level of these stop codons differed by a factor of 10 in the Weak1 context (Table 1). We revealed that the efficiency of stop codon readthrough fits into an exponential curve for both Standard and Weak1 contexts. Thus, it is a property of the stop codons themselves, regardless of the 3′ context. It is difficult to determine the cause of such an exponential dependence; obviously, it reflects a much more efficient decoding of the UGA stop codon by the suppressor or near-cognate tRNAs.Table 1Ratio of readthrough efficiencymRNAMean ± SEMRatio toUAAUAGUGAWeak1StandardUAA Standard0.5 ± 1.0–0.160.020.09–UAG Standard3.2 ± 0.66.32–0.110.31–UGA Standard28.3 ± 2.555.688.81–0.49–UAA Weak15.6 ± 0.6–0.550.10–11.01UAG Weak110.2 ± 1.01.83–0.18–3.18UGA Weak157.5 ± 2.810.295.63––2.03UAG Dyst3.7 ± 0.4––––1.17UAG TMV7.6 ± 0.4––––2.36UGA Weak256.0 ± 3.2––––1.98UGA Weak348.9 ± 4.7––––1.73 Open table in a new tab Regarding the influence of the sequence of the 3′ stop codon context, we also found significant differences in readthrough efficiency for the same codons in different contexts (Fig. 2A and Table 1). However, in this case, the difference was smaller than that between different stop codons in the same context. Thus, the readthrough of the UAA stop codon in the Standard context was 10 times worse than that in the Weak1 context, and the readthrough of the UGA stop codon was 2 times worse. Thus, the main factors determining readthrough efficiency are the type of stop codon and the sequence of the 3′ context. The influence of the type of stop codon on readthrough is likely determined by the availability of suppressor or near-cognate tRNAs in the cell and by the ability of tRNA to recognize stop codons successfully. Thus, UAA and UAG codons, in addition to suppressor tRNAs, can be recognized by glutamine or tyrosine tRNA, whereas the UGA codon can be recognized by cysteine and tryptophan tRNAs (48Beznosková P. Bidou L. Namy O. Valášek L.S. Increased expression of tryptophan and tyrosine tRNAs elevates
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