Journal of Biological Chemistry · 1999 · 257 citations · 15 references
Life SciencesBiochemistryMolecular BiologyGene ExpressionGene RegulationProtein TranslationPolyadenylation SignalsRna PolyadenylationTranslation FactorsTranslation In EukaryotesTranslation RegulationTranslation TerminationEukaryotic Elongation FactorsMessenger Rna PolyadenylationMrna Stability ControlPoly(a) Tail RegulationPoly(a)-tail-dependent TranslationPolyadenylation Signal MotifsTranslation Termination RegulationYeast Two-hybrid Screening
The mammalian GTP-binding protein GSPT, whose carboxyl-terminal sequence is homologous to the eukaryotic elongation factor EF1α, binds to the polypeptide chain releasing factor eRF1 to function as eRF3 in the translation termination. The amino-terminal domain of GSPT was, however, not required for the binding. Search for other GSPT-binding proteins in yeast two-hybrid screening system resulted in the identification of a cDNA encoding polyadenylate-binding protein (PABP), whose amino terminus is associating with the poly(A) tail of mRNAs presumably for their stabilization. The interaction appeared to be mediated through the carboxyl-terminal domain of PABP and the amino-terminal region of GSPT. Interestingly, multimerization of PABP with poly(A), which is ascribed to the action of its carboxyl-terminal domain, was completely inhibited by the interaction with the amino-terminal domain of GSPT. These results indicate that GSPT/eRF3 may play important roles not only in the termination of protein synthesis but also in the regulation of mRNA stability. Thus, the present study is the first report showing that GSPT/eRF3 carries the translation termination signal to 3′-poly(A) tail ubiquitously present in eukaryotic mRNAs. The mammalian GTP-binding protein GSPT, whose carboxyl-terminal sequence is homologous to the eukaryotic elongation factor EF1α, binds to the polypeptide chain releasing factor eRF1 to function as eRF3 in the translation termination. The amino-terminal domain of GSPT was, however, not required for the binding. Search for other GSPT-binding proteins in yeast two-hybrid screening system resulted in the identification of a cDNA encoding polyadenylate-binding protein (PABP), whose amino terminus is associating with the poly(A) tail of mRNAs presumably for their stabilization. The interaction appeared to be mediated through the carboxyl-terminal domain of PABP and the amino-terminal region of GSPT. Interestingly, multimerization of PABP with poly(A), which is ascribed to the action of its carboxyl-terminal domain, was completely inhibited by the interaction with the amino-terminal domain of GSPT. These results indicate that GSPT/eRF3 may play important roles not only in the termination of protein synthesis but also in the regulation of mRNA stability. Thus, the present study is the first report showing that GSPT/eRF3 carries the translation termination signal to 3′-poly(A) tail ubiquitously present in eukaryotic mRNAs. The yeast GSPT gene, whose product is a GTP-binding protein structurally related to the translation elongation factor EF1α, was first isolated based on the ability to complement a temperature-sensitive gst1 mutation of Saccharomyces cerevisiae (1Kikuchi Y. Shimatake H. Kikuchi A. EMBO J. 1988; 7: 1175-1182Crossref PubMed Scopus (103) Google Scholar). Because DNA synthesis was substantially arrested at the nonpermissive temperature in this mutant, the GSPTgene product appears to play an essential role at the G1 to S phase transition in the yeast cell cycle. On the other hand,SUP35 was cloned by another group investigating omnipotent suppressor mutant of S. cerevisiae, and the gene turned out to be identical to GSPT (2Kushnirov V.V. Ter-Avanesyan M.D. Telckov M.V. Surguchov A.P. Smirnov V.N. Inge-Vechtomov S.G. Gene (Amst.). 1988; 66: 45-54Crossref PubMed Scopus (168) Google Scholar). Omnipotent suppressor is a class of nonsense suppressors that is recessive and effective toward three types of nonsense codons (3Surguchov A.P. Trends Biochem. Sci. 1988; 13: 120-123Abstract Full Text PDF PubMed Scopus (7) Google Scholar). Mutations in theGSPT/SUP35 gene were shown to increase the level of translational ambiguity (4Surguchov A.P. Beretetskaya Y.V. Fominykch E.S. Pospelova E.M. Smirnov V.N. Ter-Avanesyan M.D. Inge-Vechtomov S.G. FEBS Lett. 1980; 111: 175-178Crossref PubMed Scopus (35) Google Scholar, 5Eustice D.C. Wakem L.P. Wilhelm J.M. Sherman F. J. Mol. Biol. 1986; 188: 207-214Crossref PubMed Scopus (60) Google Scholar), suggesting that this gene product may also function as a positive regulator of translational accuracy in yeast.In eukaryotic protein synthesis, all three types of termination codons are directly recognized by a polypeptide chain releasing factor, eRF1, to release synthesized polypeptide chain from ribosome, and another releasing factor, eRF3, appears to be essentially required for the ribosomal binding of eRF1. Recently, it was reported in S. cerevisiae (6Stansfield I. Jones K.M. Kushnirov V.V. Dagkesamanskaya A.R. Poznyakovski A.I. Paushkin S.V. Nierras C.R. Cox B.S. Ter-Avanesyan M.D. Tuite M.F. EMBO J. 1995; 14: 4365-4373Crossref PubMed Scopus (423) Google Scholar) and Xenopus laevis (7Zhouravleva G. Frolova L. Le Goff X. Le Guellec R. Inge-Vechtomov S. Kisselev L. Philippe M. EMBO J. 1995; 14: 4065-4072Crossref PubMed Scopus (514) Google Scholar) that the product of the GSPT/SUP35 gene forms a complex with eRF1 to function as eRF3. We previously cloned a human homologue of the yeastGSPT gene (8Hoshino S. Miyazawa H. Enomoto T. Hanaoka F. Kikuchi Y. Kikuchi A. Ui M. EMBO J. 1989; 8: 3807-3814Crossref PubMed Scopus (105) Google Scholar) and more recently isolated two mouse GSPT genes, the counterpart of human GSPT1 and a novel member of the GSPT gene family, GSPT2 (9Hoshino S. Imai M. Mizutani M. Kikuchi Y. Hanaoka F. Ui M. Katada T. J. Biol. Chem. 1998; 273: 22254-22259Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The mammalian GSPT1 and GSPT2 could also associate with eRF1 to function as eRF3, although the two GSPTs were clearly distinct from each other in terms of their amino-terminal sequences, the expression during cell cycle progression, and tissue distribution (9Hoshino S. Imai M. Mizutani M. Kikuchi Y. Hanaoka F. Ui M. Katada T. J. Biol. Chem. 1998; 273: 22254-22259Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). It thus appears that there is a functional conservation of this family from yeast to mammals in terms of translation termination. In this report, we present evidence that GSPTs may have another function by interacting with PABP that binds to the 3′-poly(A) tail of eukaryotic mRNAs. Our present results indicate that GSPT/eRF3 may play important roles not only in the termination of protein synthesis but also in the regulation of mRNA stability. The yeast GSPT gene, whose product is a GTP-binding protein structurally related to the translation elongation factor EF1α, was first isolated based on the ability to complement a temperature-sensitive gst1 mutation of Saccharomyces cerevisiae (1Kikuchi Y. Shimatake H. Kikuchi A. EMBO J. 1988; 7: 1175-1182Crossref PubMed Scopus (103) Google Scholar). Because DNA synthesis was substantially arrested at the nonpermissive temperature in this mutant, the GSPTgene product appears to play an essential role at the G1 to S phase transition in the yeast cell cycle. On the other hand,SUP35 was cloned by another group investigating omnipotent suppressor mutant of S. cerevisiae, and the gene turned out to be identical to GSPT (2Kushnirov V.V. Ter-Avanesyan M.D. Telckov M.V. Surguchov A.P. Smirnov V.N. Inge-Vechtomov S.G. Gene (Amst.). 1988; 66: 45-54Crossref PubMed Scopus (168) Google Scholar). Omnipotent suppressor is a class of nonsense suppressors that is recessive and effective toward three types of nonsense codons (3Surguchov A.P. Trends Biochem. Sci. 1988; 13: 120-123Abstract Full Text PDF PubMed Scopus (7) Google Scholar). Mutations in theGSPT/SUP35 gene were shown to increase the level of translational ambiguity (4Surguchov A.P. Beretetskaya Y.V. Fominykch E.S. Pospelova E.M. Smirnov V.N. Ter-Avanesyan M.D. Inge-Vechtomov S.G. FEBS Lett. 1980; 111: 175-178Crossref PubMed Scopus (35) Google Scholar, 5Eustice D.C. Wakem L.P. Wilhelm J.M. Sherman F. J. Mol. Biol. 1986; 188: 207-214Crossref PubMed Scopus (60) Google Scholar), suggesting that this gene product may also function as a positive regulator of translational accuracy in yeast. In eukaryotic protein synthesis, all three types of termination codons are directly recognized by a polypeptide chain releasing factor, eRF1, to release synthesized polypeptide chain from ribosome, and another releasing factor, eRF3, appears to be essentially required for the ribosomal binding of eRF1. Recently, it was reported in S. cerevisiae (6Stansfield I. Jones K.M. Kushnirov V.V. Dagkesamanskaya A.R. Poznyakovski A.I. Paushkin S.V. Nierras C.R. Cox B.S. Ter-Avanesyan M.D. Tuite M.F. EMBO J. 1995; 14: 4365-4373Crossref PubMed Scopus (423) Google Scholar) and Xenopus laevis (7Zhouravleva G. Frolova L. Le Goff X. Le Guellec R. Inge-Vechtomov S. Kisselev L. Philippe M. EMBO J. 1995; 14: 4065-4072Crossref PubMed Scopus (514) Google Scholar) that the product of the GSPT/SUP35 gene forms a complex with eRF1 to function as eRF3. We previously cloned a human homologue of the yeastGSPT gene (8Hoshino S. Miyazawa H. Enomoto T. Hanaoka F. Kikuchi Y. Kikuchi A. Ui M. EMBO J. 1989; 8: 3807-3814Crossref PubMed Scopus (105) Google Scholar) and more recently isolated two mouse GSPT genes, the counterpart of human GSPT1 and a novel member of the GSPT gene family, GSPT2 (9Hoshino S. Imai M. Mizutani M. Kikuchi Y. Hanaoka F. Ui M. Katada T. J. Biol. Chem. 1998; 273: 22254-22259Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The mammalian GSPT1 and GSPT2 could also associate with eRF1 to function as eRF3, although the two GSPTs were clearly distinct from each other in terms of their amino-terminal sequences, the expression during cell cycle progression, and tissue distribution (9Hoshino S. Imai M. Mizutani M. Kikuchi Y. Hanaoka F. Ui M. Katada T. J. Biol. Chem. 1998; 273: 22254-22259Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). It thus appears that there is a functional conservation of this family from yeast to mammals in terms of translation termination. In this report, we present evidence that GSPTs may have another function by interacting with PABP that binds to the 3′-poly(A) tail of eukaryotic mRNAs. Our present results indicate that GSPT/eRF3 may play important roles not only in the termination of protein synthesis but also in the regulation of mRNA stability.
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A novel genetic system to detect protein–protein interactions
Stanley Fields, Ok-Kyu Song · Nature · 1989 · 6.1K citations