Nascent Peptide-dependent Translation Arrest Leads to Not4p-mediated Protein Degradation by the Proteasome

The potentially deleterious effects of aberrant mRNA lacking a termination codon (nonstop mRNA) are ameliorated by translation arrest, proteasome-mediated protein destabilization, and rapid mRNA degradation. Because polylysine synthesis via translation of the poly(A) mRNA tail leads to translation arrest and protein degradation by the proteasome, we examined the effects of other amino acid sequences. Insertion of 12 consecutive basic amino acids between GFP and HIS3 reporter genes, but not a stem-loop structure, resulted in degradation of the truncated green fluorescent protein (GFP) products by the proteasome. Translation arrest products derived from GFP-R12-FLAG-HIS3 or GFP-K12-FLAG-HIS3 mRNA were detected in a not4Δ mutant, and MG132 treatment did not affect the levels of the truncated arrest products. Deletion of other components of the Ccr4-Not complex did not increase the levels of the translation arrest products or reporter mRNAs. A L35A substitution in the Not4p RING finger domain, which disrupted its interaction with the Ubc4/Ubc5 E2 enzyme and its activity as an ubiquitin-protein ligase, also abrogated the degradation of arrest products. These results suggest that Not4p, a component of the Ccr4-Not complex, may act as an E3 ubiquitin-protein ligase for translation arrest products. The results let us propose that the interaction between basic amino acid residues and the negatively charged exit tunnel of the ribosome leads to translation arrest followed by Not4p-mediated ubiquitination and protein degradation by the proteasome. The potentially deleterious effects of aberrant mRNA lacking a termination codon (nonstop mRNA) are ameliorated by translation arrest, proteasome-mediated protein destabilization, and rapid mRNA degradation. Because polylysine synthesis via translation of the poly(A) mRNA tail leads to translation arrest and protein degradation by the proteasome, we examined the effects of other amino acid sequences. Insertion of 12 consecutive basic amino acids between GFP and HIS3 reporter genes, but not a stem-loop structure, resulted in degradation of the truncated green fluorescent protein (GFP) products by the proteasome. Translation arrest products derived from GFP-R12-FLAG-HIS3 or GFP-K12-FLAG-HIS3 mRNA were detected in a not4Δ mutant, and MG132 treatment did not affect the levels of the truncated arrest products. Deletion of other components of the Ccr4-Not complex did not increase the levels of the translation arrest products or reporter mRNAs. A L35A substitution in the Not4p RING finger domain, which disrupted its interaction with the Ubc4/Ubc5 E2 enzyme and its activity as an ubiquitin-protein ligase, also abrogated the degradation of arrest products. These results suggest that Not4p, a component of the Ccr4-Not complex, may act as an E3 ubiquitin-protein ligase for translation arrest products. The results let us propose that the interaction between basic amino acid residues and the negatively charged exit tunnel of the ribosome leads to translation arrest followed by Not4p-mediated ubiquitination and protein degradation by the proteasome. Cells have surveillance systems that recognize and eliminate aberrant mRNAs to prevent the production of potentially harmful protein products. In eukaryotes, an aberrant mRNA lacking a termination codon (nonstop mRNA) may be recognized and eliminated by the quality control system referred to as nonstop decay (1van Hoof A. Frischmeyer P.A. Dietz H.C. Parker R. Science.. 2002; 295: 2262-2264Google Scholar, 2Frischmeyer P.A. van Hoof A. O'Donnell K. Guerrerio A.L. Parker R. Dietz H.C. Science.. 2002; 295: 2258-2261Google Scholar). It has been shown that nonstop mRNA is rapidly degraded by a 3′-to-5′ degradation pathway in yeast (1van Hoof A. Frischmeyer P.A. Dietz H.C. Parker R. Science.. 2002; 295: 2262-2264Google Scholar). We have shown that translation of nonstop mRNA is repressed after the initiation step (3Inada T. Aiba H. EMBO J... 2005; 24: 1584-1595Google Scholar, 4Ito-Harashima S. Kuroha K. Tatematsu T. Inada T. Genes Dev... 2007; 21: 519-524Google Scholar); the level of the product of nonstop mRNA containing a poly(A) tail was reduced by ∼100-fold, and this reduction was due to rapid mRNA degradation, translation repression, and protein destabilization, at least in part, by the proteasome (3Inada T. Aiba H. EMBO J... 2005; 24: 1584-1595Google Scholar, 4Ito-Harashima S. Kuroha K. Tatematsu T. Inada T. Genes Dev... 2007; 21: 519-524Google Scholar). Interestingly, insertion of a poly(A) tract upstream of a termination codon resulted in translation repression and protein destabilization but not rapid mRNA decay (3Inada T. Aiba H. EMBO J... 2005; 24: 1584-1595Google Scholar, 4Ito-Harashima S. Kuroha K. Tatematsu T. Inada T. Genes Dev... 2007; 21: 519-524Google Scholar). Recently, it has been shown that translation of nonstop mRNA also is repressed after initiation in mammalian cells and that the ribosome stalls on poly(A) sequences, a common modification of eukaryotic mRNA that is not normally translated (5Akimitsu N. Tanaka J. Pelletier J. EMBO J... 2007; 26: 2327-2338Google Scholar). Therefore, translation repression following polylysine synthesis may be conserved among eukaryotes. We propose that ribosomes recognize the translation of poly(A) tails as an aberrant process, resulting in translation arrest and degradation of the protein product by the proteasome. Translation elongation is an important step of gene regulation; the expression levels of several genes are known to be regulated by translation arrest. In prokaryotes, the regulation of the secM gene, for example, has been extensively analyzed. The interaction between the SecM nascent peptide and the wall of the ribosomal exit tunnel functions as a discriminating gate during translation (6Nakatogawa H. Ito K. Mol. Cell.. 2001; 7: 185-192Google Scholar, 7Nakatogawa H. Ito K. Cell.. 2002; 108: 629-636Google Scholar). Several eukaryotic examples of translation arrest also have been described (8Onouchi H. Lambein I. Sakurai R. Suzuki A. Chiba Y. Naito S. Biochem. Soc. Trans... 2004; 32: 597-600Google Scholar, 9Chiba Y. Ishikawa M. Kijima F. Tyson R.H. Kim J. Yamamoto A. Nambara E. Leustek T. Wallsgrove R.M. Naito S. Science.. 1999; 286: 1371-1374Google Scholar). Moreover, the synthesis of polylysine caused by translation of a poly(A) sequence leads to translation arrest (3Inada T. Aiba H. EMBO J... 2005; 24: 1584-1595Google Scholar, 4Ito-Harashima S. Kuroha K. Tatematsu T. Inada T. Genes Dev... 2007; 21: 519-524Google Scholar). Recent measurement of the electrostatic potential of the ribosome exit tunnel using chemical modification methods revealed that the wall of the tunnel is negatively charged in eukaryotes (10Lu J. Kobertz W.R. Deutsch C. J. Mol. Biol... 2007; 371: 1378-1391Google Scholar, 11Lu J. Deutsch C. Nat. Struct. Mol. Biol... 2005; 12: 1123-1129Google Scholar). These results suggest that consecutive positively charged amino acid residues in a nascent protein may have a high affinity for the negatively charged ribosomal exit tunnel. A more recent report demonstrated that positively charged residues cause pausing in mammalian ribosomes in vitro and showed how far the positively charged side chains move into the tunnel (12Lu J. Deutsch C. J. Mol. Biol... 2008; 384: 73-86Google Scholar). The evolutionarily conserved Ccr4-Not complex regulates mRNA biosynthesis at various steps. First, Ccr4p/Caf1p has a deadenylase activity that functions in mRNA deadenylation (13Tucker M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J... 2002; 21: 1427-1436Google Scholar, 14Chen J. Rappsilber J. Chiang Y.C. Russell P. Mann M. Denis C.L. J. Mol. Biol... 2001; 314: 683-694Google Scholar-15Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell.. 2001; 104: 377-386Google Scholar). Second, Ccr4p/Caf1p regulates histone H3K4 trimethylation (H3K4me3), which plays a significant role in chromatin organization, gene transcription, and epigenetic regulation via interactions with the proteasome to regulate its recruitment to genes (16Laribee R.N. Shibata Y. Mersman D.P. Collins S.R. Kemmeren P. Roguev A. Weissman J.S. Briggs S.D. Krogan N.J. Strahl B.D. Proc. Natl. Acad. Sci. U. S. A... 2007; 104: 5836-5841Google Scholar). In yeast, the Ccr4-Not core complex is composed of nine components. The Not1–5 proteins are critical for transcriptional repression. It recently was shown that NOT1, NOT2, NOT4, and NOT5 are required for trimethylation of H3K4 (16Laribee R.N. Shibata Y. Mersman D.P. Collins S.R. Kemmeren P. Roguev A. Weissman J.S. Briggs S.D. Krogan N.J. Strahl B.D. Proc. Natl. Acad. Sci. U. S. A... 2007; 104: 5836-5841Google Scholar, 17Mulder K.W. Brenkman A.B. Inagaki A. van den Broek N.J. Timmers H.T. Nucleic Acids Res... 2007; 35: 2428-2439Google Scholar). Third, the Not4p subunit of the Ccr4-Not complex has an E3 3The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; E2, ubiquitin carrier protein; GFP, green fluorescent protein; DIG, digoxigenin. ubiquitin-protein ligase activity for the conserved ribosome-associated heterodimeric EGD complex. This complex consists of the Egd1p and Egd2p subunits in yeast and is named NAC (nascent polypeptide-associated complex) in mammals. Finally, the Not4p displays an Ubc4p-dependent ubiquitination activity in vitro (18Mulder K.W. Inagaki A. Cameroni E. Mousson F. Winkler G.S. De Virgilio C. Collart M.A. Timmers H.T. Genetics.. 2007; 176: 181-192Google Scholar, 19Panasenko O. Landrieux E. Feuermann M. Finka A. Paquet N. Collart M.A. J. Biol. Chem... 2006; 281: 31389-31398Google Scholar), which depends on the RING finger domain of Not4p (18Mulder K.W. Inagaki A. Cameroni E. Mousson F. Winkler G.S. De Virgilio C. Collart M.A. Timmers H.T. Genetics.. 2007; 176: 181-192Google Scholar). In this study, we show that 12 consecutive basic amino acid residues cause translation arrest followed by cotranslational protein degradation by the proteasome. In contrast, a stem-loop structure induces only translation arrest but not degradation of the arrested product. Not4p is required for the degradation of translation arrest products, although Not3p and Not5p of the Ccr4-Not complex are not. In addition, translation arrest products are not degraded by the proteasome in the not4L35A mutant, in which ubiquitination of the EGD complex and the interaction with E2 enzymes are defective. These results strongly suggest that nascent peptide sequences play crucial roles in translation elongation arrest, leading to Not4p-mediated protein degradation by the proteasome. More general roles of endogenous nascent peptides in the regulation of translation and protein degradation also are discussed. Strains and General Methods—Standard procedures were used to manipulate yeast cells (20Amberg D.C. Burke D.J. Strathern J.N. Methods in 2005; Scholar). The yeast and used in this are described in of NOT4, and NOT5 were using a A. D.J. A. A. P. Scholar). procedures were as described J. Russell The from Scholar). were and into the of to the which sequences various acid or a stem-loop structure between GFP and A was in a using and and into to a was using and were and into the of to of the cells were on and proteins from to were The products derived from the reporter genes were detected using with The used for were and The recognize the chains of proteins but not ubiquitin or the protein M. H. H. Scholar). The of the were with the system using and the levels of the products were on a with a the of the was the of the a of was The of the for the were with from the and the levels of the products in the with the control products were was using in the of and mRNA was using and for acid to the were with the following HIS3 and and GFP and The were using the system and and cells were at and by The cells were with and were as described T. E. D. A.B. 2002; Scholar). The of was in in using a were on of the and at in a for at The were were with measurement at using a to a were and for as described by the was that insertion of a poly(A) tract upstream of a termination codon results in translation repression and protein destabilization but not rapid mRNA decay (3Inada T. Aiba H. EMBO J... 2005; 24: 1584-1595Google Scholar, 4Ito-Harashima S. Kuroha K. Tatematsu T. Inada T. Genes Dev... 2007; 21: 519-524Google Scholar). translation is arrested by other amino acid sequences and the translation arrest product is degraded by the proteasome, we various amino acid sequences between the GFP and HIS3 genes We that translation may be arrested the ribosome and the translation arrest product be detected as a truncated GFP product in the of a proteasome the level of the product was of the control and in the of a translation arrest product was detected as a truncated GFP product and We also that insertion of 12 consecutive residues strongly reduced the level of the product of the control and The is to the for an the sequence and more following the This eliminated translation repression and that the basic amino acid sequence plays a crucial role for translation repression. was a between the translation arrest of and The mRNA levels of and were that the sequence play a role in translation repression. is that may to poly(A) sequences and translation that MG132 the we with which recognize and but not shown in the levels of proteins were in the of In addition, we that the levels of the truncated products derived from in the of MG132 in which show an of MG132 A.L. J. Biol. Chem... Scholar). We also that the levels and of the mRNAs derived from or GFP-R12-FLAG-HIS3 were not by MG132 that the truncated proteins were not from truncated mRNAs. We that the levels of the mRNA and protein derived from were derived from the control reporter and did not affect level of mRNA and protein derived from and and Insertion of but not acid reduced the levels of the products, the effects of polylysine product derived from the acid and was not in the of These results suggest that basic amino acid residues may degradation of arrest products. We also examined nascent peptides are degraded by the proteasome translation is arrested by a stem-loop The level of the product from mRNA was reduced of the control and the mRNA level was not reduced and These results that translation is arrested by stem-loop as described K. M. J. Mol. 2004; Scholar). MG132 treatment did not affect the expression of the products from translation arrest caused by the stem-loop structure and This strongly that nascent not translation arrest by a in cotranslational protein degradation by the proteasome. the truncated products derived from GFP-K12-FLAG-HIS3 or GFP-R12-FLAG-HIS3 may from after translation is the proteins after of translation were examined using The levels of the products were not as a of after the of that the products were as as MG132 treatment did not increase the levels of or These results suggest that the truncated products derived from and GFP-R12-FLAG-HIS3 were not by degradation or the of products and are with a in which the truncated product detected in the of MG132 is by translation arrest. we propose that nascent peptides translation arrest, which in leads to cotranslational protein degradation by the proteasome in for Translation and the of the amino acid sequences required to translation arrest and cotranslational protein degradation, we examined the expression of containing sequences that other acid sequences between GFP and in the or of a proteasome We significant in the levels of the proteins containing 12 although truncated GFP products derived from reporter genes were not after MG132 treatment and In contrast, truncated products derived from were detected only the of MG132 and although the levels were not as high as by 12 consecutive or of a abrogated the arrest product in the of MG132 and These results suggest that translation of consecutive 12 basic or residues results in translation arrest, which leads to nascent protein degradation by the proteasome. We also truncated products from the which of the sequence and polylysine and The levels of truncated products in the of MG132 and a production of truncated products and These results that are required for the translation arrest with protein degradation as described S. Kuroha K. Tatematsu T. Inada T. Genes Dev... 2007; 21: 519-524Google Scholar). We also detected several truncated GFP products derived from The level of was to that of control product and In addition, the levels of truncated products did not increase in the of MG132 and These results strongly suggest that the truncated GFP products are not translation arrest products. We also detected a truncated GFP product derived from MG132 treatment and A truncated product was detected using with and its level to that of the product was to the results for truncated GFP not These suggest that is of the proteasome MG132 on the levels of truncated product and that 12 consecutive basic amino acid residues translation arrest, we a reporter that a sequence between GFP and the sequence did not The truncated product was only in the of MG132 and These results strongly suggest that consecutive basic amino acids or polylysine or sequences are required for translation arrest with protein degradation. Not4p for of Translation results that nascent protein degradation is that an E3 ubiquitin-protein ligase may be with the Not4p, a component of the Ccr4-Not complex, has been as an E3 ubiquitin-protein ligase for EGD with nascent O. Landrieux E. Feuermann M. Finka A. Paquet N. Collart M.A. J. Biol. Chem... 2006; 281: 31389-31398Google Scholar). Therefore, we the expression from GFP-R12-FLAG-HIS3 in a not4Δ In the not4Δ mutant, the level of the truncated product derived from GFP-R12-FLAG-HIS3 was with the level of GFP-R12-FLAG-HIS3 was not by the of MG132 and A truncated mRNA was not in the of MG132 We also that the levels of truncated products derived from or also were in the not4Δ and These results strongly suggest that Not4p is required for the degradation of translation arrest products by the proteasome. The level of GFP-R12-FLAG-HIS3 mRNA also in the not4Δ and although the level of the protein was and that translation arrest of GFP-R12-FLAG-HIS3 mRNA may be in the not4Δ Because Not4p is in transcriptional regulation via H3K4 the in the not4Δ may be this we the levels of proteins using with It was shown that is a component of the of the proteasome and that the activity of the proteasome is in the E. N. N. Y. J. Biol. Chem... 2005; Scholar). We that the levels of proteins were in the with the not4Δ that the activity of the proteasome in the was more in the not4Δ The increase in the truncated GFP product in the mutant, was in the not4Δ The truncated product in the and also was not and We results for the mRNA levels and Because Not4p and are required for trimethylation of H3K4 (16Laribee R.N. Shibata Y. Mersman D.P. Collins S.R. Kemmeren P. Roguev A. Weissman J.S. Briggs S.D. Krogan N.J. Strahl B.D. Proc. Natl. Acad. Sci. U. S. A... 2007; 104: 5836-5841Google Scholar, 17Mulder K.W. Brenkman A.B. Inagaki A. van den Broek N.J. Timmers H.T. Nucleic Acids Res... 2007; 35: 2428-2439Google Scholar), results suggest that the in transcriptional regulation in the not4Δ may not for the levels of the truncated product. These results are with the that Not4p is required for the degradation of truncated products by the proteasome. The domain of Not4p a RING finger domain, which its interaction with the E2 ligase the L35A disrupted the ubiquitin-protein ligase activity of Not4p (18Mulder K.W. Inagaki A. Cameroni E. Mousson F. Winkler G.S. De Virgilio C. Collart M.A. Timmers H.T. Genetics.. 2007; 176: 181-192Google Scholar). The level of the truncated product derived from was in the not4Δ the but not in the not4Δ the Interestingly, was in the of that Not4p may be with Not4p to as an E3 ubiquitin-protein ligase for translation arrest products, which is followed by proteasome-mediated degradation. of mRNA demonstrated that translation of a poly(A) tail from nonstop mRNA results in translation arrest and protein degradation of the nonstop products (3Inada T. Aiba H. EMBO J... 2005; 24: 1584-1595Google Scholar, 4Ito-Harashima S. Kuroha K. Tatematsu T. Inada T. Genes Dev... 2007; 21: 519-524Google Scholar). the role of Not4p in the degradation of nonstop products, we examined the expression of nonstop products in a not4Δ The level of the nonstop product derived from did not increase in the not4Δ and that Not4p is not in the degradation of nonstop products. We also that the levels of and mRNA were not in the not4Δ although the level of mRNA was that of mRNA as described S. Kuroha K. Tatematsu T. Inada T. Genes Dev... 2007; 21: 519-524Google Scholar). This is with the role of Not4p as an E3 ubiquitin-protein ligase for translation arrest products but not for aberrant proteins from the mRNA Translation with results suggest that translation arrest by sequences consecutive basic amino acid residues leads to Not4p-mediated protein degradation by the proteasome. a role for translation arrest in gene we sequences for consecutive basic amino acid sequences and genes The endogenous sequences were between GFP and HIS3 to translation arrest and protein degradation products derived from the reporter genes were detected in the of The of translation arrest and protein degradation was with the sequence derived from and products derived from the reporter genes were also detected in the not4Δ mutant, and the sequence showed the translation arrest activity A role for sequences in gene regulation is Translation and by regulation by the synthesis of amino acid sequences and translation arrest has been in and eukaryotes. Translation arrest of secM mRNA is with protein and plays a crucial role in the of protein expression H. Ito K. Cell.. 2002; 108: 629-636Google Scholar, H. A. Ito K. 2004; 7: Scholar). The SecM nascent peptide with the exit tunnel of the ribosome at the the translation elongation of secM mRNA (6Nakatogawa H. Ito K. Mol. Cell.. 2001; 7: 185-192Google Scholar, 7Nakatogawa H. Ito K. Cell.. 2002; 108: 629-636Google Scholar). We that 12 consecutive basic residues caused translation arrest, that positively charged side chains with the ribosome tunnel by ribosomal N. P. J. M. 1999; Scholar). The exit tunnel wall of the mammalian ribosome has a electrostatic potential (10Lu J. Kobertz W.R. Deutsch C. J. Mol. Biol... 2007; 371: 1378-1391Google and may with peptides that positively charged residues (12Lu J. Deutsch C. J. Mol. Biol... 2008; 384: 73-86Google Scholar). The interaction for the consecutive positively charged side chains has to be although the is a Recently, it was shown that translation of nonstop mRNA is repressed after initiation and that the ribosome stalls on poly(A) sequences in mammalian cells (5Akimitsu N. Tanaka J. Pelletier J. EMBO J... 2007; 26: 2327-2338Google Scholar). In addition, translation of poly(A) mRNA was in an in vitro system using (5Akimitsu N. Tanaka J. Pelletier J. EMBO J... 2007; 26: 2327-2338Google Scholar). Therefore, translation repression by consecutive basic amino acids may be conserved among eukaryotes. of Translation on that Not4p is required for the degradation of translation arrest products. Not4p is also in transcriptional regulation via trimethylation of H3K4 (16Laribee R.N. Shibata Y. Mersman D.P. Collins S.R. Kemmeren P. Roguev A. Weissman J.S. Briggs S.D. Krogan N.J. Strahl B.D. Proc. Natl. Acad. Sci. U. S. A... 2007; 104: 5836-5841Google Scholar, 17Mulder K.W. Brenkman A.B. Inagaki A. van den Broek N.J. Timmers H.T. Nucleic Acids Res... 2007; 35: 2428-2439Google Scholar). Therefore, that may play a crucial role in the of an activity that may be in the not4Δ mutant, leading to the of translation arrest products. of not this First, we that the levels of proteins were in the with the not4Δ mutant, although the level of the truncated GFP product was that in the not4Δ Second, translation of nonstop mRNA was not in the not4Δ although the nonstop product was degraded by the proteasome (3Inada T. Aiba H. EMBO J... 2005; 24: 1584-1595Google Scholar, 4Ito-Harashima S. Kuroha K. Tatematsu T. Inada T. Genes Dev... 2007; 21: 519-524Google Scholar). These suggest that the reduced degradation of translation arrest products in the not4Δ is not due to a general in We that translation arrest by a stem-loop not to arrest product degradation by the proteasome This is with the that nascent peptide sequences are required for cotranslational degradation of arrest products by the proteasome. is the cotranslational degradation of the translation arrest is that the ribosome may have a high affinity for Not4p, leading to ubiquitination of the nascent It has been shown that a SecM nascent peptide the of the ribosome K. J. A. Nat. Struct. Mol. Biol... 2006; Scholar), that the interaction between a nascent peptide and the exit tunnel wall regulate translation by the of the We that interactions between nascent peptides and the exit tunnel wall to in the of the ribosome and increase between the ribosome and Not4p, protein degradation. It was shown that the interaction between Not4p and which is on the Not4p RING finger domain, is required for the ubiquitin-protein ligase activity for the EGD complex (18Mulder K.W. Inagaki A. Cameroni E. Mousson F. Winkler G.S. De Virgilio C. Collart M.A. Timmers H.T. Genetics.. 2007; 176: 181-192Google Scholar, 19Panasenko O. Landrieux E. Feuermann M. Finka A. Paquet N. Collart M.A. J. Biol. Chem... 2006; 281: 31389-31398Google Scholar). has recently been shown to with the proteasome in to (18Mulder K.W. Inagaki A. Cameroni E. Mousson F. Winkler G.S. De Virgilio C. Collart M.A. Timmers H.T. Genetics.. 2007; 176: 181-192Google Scholar). from this suggest that translation arrest by consecutive basic amino acids leads to Not4p-mediated protein degradation by the proteasome. we propose a more general in which translation arrest caused by nascent peptides leads to cotranslational protein degradation by the proteasome. Not4p is with ribosomes and may be in the ubiquitination of aberrant nascent on ribosomes by the E2 enzymes and as as the proteasome. Translation by results of this us to that nascent peptides may play more general roles in translation protein and protein by the ribosome We that the sequence from translation arrest is a component of the K. S. J. Biol. Chem... 2005; Scholar), an that to degradation of mRNA T. T. J. S. Mol. Biol... 2004; 24: Scholar). Because translation arrest leads to of mRNA in and eukaryotes (8Onouchi H. Lambein I. Sakurai R. Suzuki A. Chiba Y. Naito S. Biochem. Soc. Trans... 2004; 32: 597-600Google Scholar, 9Chiba Y. Ishikawa M. Kijima F. Tyson R.H. Kim J. Yamamoto A. Nambara E. Leustek T. Wallsgrove R.M. Naito S. Science.. 1999; 286: 1371-1374Google Scholar, Parker R. 2006; Scholar, H. Y. Y. M. Y. Sakurai R. N. D. Y. Naito S. Genes Dev... 2005; Scholar, T. K. H. Inada T. Aiba H. J. Biol. Chem... 2004; T. K. Yamamoto Y. Inada T. Aiba H. 2004; Scholar), translation arrest by consecutive basic amino acid sequences may to an of and may be for this It is that the products are degraded from the by an or from the by and only be detected in to this are We for the and for and We and for the yeast We also and for and We also of the for the of the and with