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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Quality control systems eliminate aberrant proteins derived from aberrant mRNAs. Two E3 ubiquitin ligases, Ltn1 and Not4, are involved in proteasomal protein degradation coupled to translation arrest. Here, we evaluated nonstop and translation arrest products degraded in a poly(A) tail-independent manner. Ltn1 was found to degrade aberrant nonstop polypeptides derived from nonstop mRNA lacking a termination codon, but not peptidyl-tRNA, even in the absence of the ribosome dissociation complex Dom34:Hbs1. The receptor for activated C kinase (RACK1/ASC1) was identified as a factor required for nascent peptide-dependent translation arrest as well as Ltn1-dependent protein degradation. Both Not4 and Ltn1 were involved in the degradation of various arrest products in a poly(A) tail-independent manner. Furthermore, carboxyl terminus-truncated degradation intermediates of arrest products were stabilized in a cdc48-3 mutant defective in unfolding or the disassembly related to proteasomal degradation. Thus, we propose that stalled ribosomes may be dissociated into subunits and that peptidyl-tRNA on the 60S subunit is ubiquitinated by Ltn1 and Cdc48 is required for the degradation following release from tRNA.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

mRNA surveillance systems recognize aberrant translation elongation and termination and induce rapid mRNA decay. In particular, recent studies have shown that aberrant proteins derived from aberrant mRNAs are recognized and rapidly degraded by specific quality control systems and that this process is crucial for repressing the expression of aberrant products. Aberrant mRNA lacking a termination codon (nonstop mRNA) is produced mainly by polyadenylation within an open reading frames (ORF) and is rapidly degraded by the quality control system designated nonstop decay (NSD) (Frischmeyer et al. 2002; van Hoof & Parker 2002). In addition to the rapid decay of nonstop mRNA by exosomes, rapid degradation of nonstop proteins is another crucial mechanism for preventing aberrant protein expression (Inada & Aiba 2005; Ito-Harashima et al. 2007; Wilson et al. 2007; Bengtson & Joazeiro 2010).

The stalling of ribosomes during translational elongation due to the formation of stable RNA secondary structures, depurination of mRNA, rare codons or premature stop codons leads to endonucleolytic cleavage of the mRNA in the vicinity of the stalled site, a process that is referred to as no-go decay (Doma & Parker 2006; Gandhi et al. 2008; Chen et al. 2010; van den Elzen et al. 2010; Kobayashi et al. 2010; Izawa et al. 2012; Tsuboi et al. 2012). Translation arrest induced by a nascent peptide with positively charged residues results in co-translational degradation of the arrested protein product by the proteasome (Dimitrova et al. 2009). Two ubiquitin ligases, Not4 and Ltn1, are involved in the degradation of the arrest product resulting from a poly-lysine sequence (Wilson et al. 2007; Dimitrova et al. 2009; Bengtson & Joazeiro 2010; Brandman et al. 2012; Defenouillere et al. 2013). Not4 is a component of the Ccr4-Not deadenylase, but is not essential for the activity and assembly of that complex (Bai et al. 1999). Not4 acts as an E3 ubiquitin ligase for the nascent peptide-associated complex (NAC) (Panasenko et al. 2006; Mulder et al. 2007). In addition, Not4 associates with polyribosomes, as revealed by polysome fractionation (Dimitrova et al. 2009; Panasenko & Collart 2012), and is involved in protein degradation of the translation arrest products produced by poly-lysine sequences, but not those of nonstop proteins (Dimitrova et al. 2009). Ltn1 is also involved in the co-translational protein degradation of arrest products produced by the translation of poly(A) sequences (Wilson et al. 2007; Bengtson & Joazeiro 2010). The mechanisms by which ubiquitin ligases recognize aberrant products produced from aberrant mRNAs have been investigated. For example, Ltn1 was shown to be involved in co-translational protein degradation of arrest products produced by the translation of poly(A) sequences, suggesting that Ltn1 may recognize the specific conformation used by a stalled ribosome when translating a poly(A) sequence (Bengtson & Joazeiro 2010). ASC1 encodes an orthologue of a receptor for activated C kinase (RACK1) in Saccharomyces cerevisiae, and it was also identified as a factor required for nascent peptide-dependent translation arrest. RACK1/ASC1-dependent translation arrest was shown to lead to co-translational degradation of the arrest product by the proteasome (Kuroha et al. 2010). These findings suggest that RACK1/ASC1 may be crucial for the recognition of aberrant polypeptides by Ltn1; however, the role of RACK1/ASC1 in Ltn1-dependent rapid degradation of aberrant products by the proteasome has not been investigated.

Ltn1 is specifically associated with the 60S ribosomal subunit, suggesting that ubiquitination of aberrant proteins may take place on 60S subunits containing peptidyl-tRNA in vivo (Bengtson & Joazeiro 2010; Brandman et al. 2012) and in vitro (Shao et al. 2013). The Dom34:Hbs1 complex stimulates the endonucleolytic cleavage of mRNA induced by translation arrest in vivo (Doma & Parker 2006; Chen et al. 2010; van den Elzen et al. 2010; Kobayashi et al. 2010) and dissociates the subunits of stalled ribosomes in vitro (Shoemaker et al. 2010; Pisareva et al. 2011; Shoemaker & Green 2011). In addition, Dom34:Hbs1 dissociates stalled ribosomes at the 3′ end of nonstop mRNA and stimulates its degradation by exosomes in vivo (Kobayashi et al. 2010; Izawa et al. 2012). However, the relationship between Dom34:Hbs1-dependent subunit dissociation of stalled ribosomes and Ltn1-dependent rapid degradation of nonstop protein products remains to be resolved.

Recent studies have identified novel factors, including Rqc1 and Tae2, involved in co-translational degradation of arrest products produced by polybasic amino-acid-sequence-inducing translation arrest (Brandman et al. 2012; Defenouillere et al. 2013). Rqc1, Tae2 and Ltn1 are members of the 60S subunit-bound complex, and 60S binding of Rqc1 is independent of Ltn1 and Tae2, whereas 60S binding of Tae2 is independent of Rqc1. Furthermore, affinity-purified Rqc1 or Tae2 proteins have been associated with the 60S subunit and ubiquitinated products, indicating that these factors stimulate but are not essential for the ubiquitination of arrest products on the 60S subunit. Rqc1 levels are auto-regulated by a negative feedback loop that depends on the basic amino acid sequence at the N-terminal region of Rqc1. In addition, Cdc48 and its cofactors, Ufd1 and Npl4, are also associated with the 60S subunit complex, and the binding of the Cdc48-Ufd1-Npl4 complex largely depends on Ltn1 and Tae2, which suggests that ubiquitinated arrest products on the 60S subunit may recruit the Cdc48 complex. Recent studies have also clearly showed that Cdc48/p97 promotes the degradation of aberrant nascent polypeptides bound to the ribosome (Brandman et al. 2012; Defenouillere et al. 2013; Verma et al. 2013).

In this study, we examined the mechanism of co-translational degradation of aberrant proteins by the proteasome, as well as the roles of two E3 ubiquitin ligases and the stalled ribosome dissociation factor Dom34:Hbs1. We found that aberrant nonstop polypeptides derived from nonstop mRNA lacking a poly(A) tail were dramatically stabilized following LTN1 deletion in the absence of the ribosome dissociation factor Dom34:Hbs1. Furthermore, both Not4 and Ltn1 were involved in the degradation of various arrest products associated with RACK1/ASC1-dependent translation arrest. Degradation intermediates of arrest products were stabilized in a cdc48-3 temperature-sensitive mutant, and the degradation intermediate was found to lack a carboxyl-terminal region. These findings suggest that aberrant proteins may be degraded from the carboxyl terminus and that unfolding of target proteins by Cdc48 is required for proteasomal degradation. Based on these results, we propose models for the protein degradation pathways associated with no-go and nonstop mRNA surveillance systems.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Ltn1 degrades products of mRNAs lacking a termination codon regardless of a poly(A) tail

To determine the substrate specificity of Ltn1, the degradation of an aberrant protein derived from a stop codon-less mRNA that lacks a poly(A) tail was examined in the ltn1 mutant. The Dom34:Hbs1 complex was required for protein synthesis from the stop codon-less GFP-Rz mRNA that was produced from the GFP-Rz-FLAG-HIS3 (Rz) reporter gene via the self-cleavage of a hammerhead ribozyme sequence inserted within the ORF (Fig. 1A) (Kobayashi et al. 2010; Izawa et al. 2012; Tsuboi et al. 2012). The amount of protein derived from GFP-Rz mRNA was significantly increased in ltn1Δ mutant cells, but not not4Δ mutant cells (Fig. 1B, lanes 9-12). As expected, the level of protein derived from GFP-FLAG-HIS3-NS (NS) mRNA, which contains a poly(A) tail, was also significantly increased in ltn1Δ mutant cells, but not in not4Δ mutant cells (Fig. 1B, lanes 5–8). These findings indicate that Ltn1 primarily degrades protein products derived from aberrant mRNAs that lack a termination codon. Furthermore, a deletion or point mutation in the RING domain of Ltn1, which decreases the binding affinity of the E2 ubiquitin-conjugating enzyme, was defective in down-regulating both nonstop proteins (Fig. 1C, lanes 5–12). We confirmed that the expression level of each protein was almost the same as that of wild type (Fig. S1 in Supporting Information), indicating that the absence of complementation in the mutants is due to loss of protein interaction and not of absence of truncated or mutated forms of these proteins. These data indicate that the E3 ubiquitin ligase activity of Ltn1 is required for the degradation of nonstop proteins and that translation of a poly(A) tail in nonstop mRNA is not essential for efficient Ltn1-dependent proteasomal degradation.

image

Figure 1. Ltn1 destabilizes nonstop protein products. (A) Schematic drawing of the reporter NS (GFP-FLAG-HIS3-NS) and Rz (GFP-Rz-FLAG-HIS3) mRNAs lacking a termination codon. The filled boxes indicate the open reading frames, the lines represent nontranslated regions, and the tract of As denotes the poly(A) tail. The dark box shows the FLAG tag sequence, and Rz indicates a hammerhead ribozyme sequence that induces self-cleavage. (B) The level of nonstop products was increased in ltn1Δ, but not not4Δ, mutant cells. Yeast strains containing the p416GPDp-GFP-FLAG-HIS3 (), p416 GPDp-GFP-FLAG-HIS3-NS (NS) or p416 GPDp-GFP-Rz-FLAG-HIS3 (Rz) reporter plasmids were analyzed by Western blotting with anti-GFP (Top panel) or anti-EF-1α antibodies (Bottom panel). When indicated, the samples were diluted 10-fold. (C) Binding of Ltn1 to the E2 enzyme is required for the down-regulation of nonstop products. W303ltn1Δ mutant cells containing the GFP-FLAG-HIS3-NS (NS) or GFP-Rz-FLAG-HIS3 (Rz) nonstop reporter genes were transformed with plasmids expressing wild-type Ltn1 or Ltn1 mutant proteins defective in binding to the E2 enzyme. The levels of arrest products were determined by Western blotting with anti-GFP (Top panel) or anti-EF-1α antibodies (Bottom panel). When indicated, the samples were diluted 10-fold. (D) Peptidyl-tRNA derived from GFP-Rz mRNA was not increased in ltn1Δ mutant cells. Cell extracts were analyzed by NuPAGE followed by Western blotting. The indicated samples were treated with RNaseA before NuPAGE. (E) Aberrant polypeptides were dramatically stabilized by LTN1 deletion in the absence of Dom34. Cell extracts were prepared from cells harboring the p416GPDp-GFP-Rz-FLAG-HIS3 plasmid, and polysome analysis followed by NuPAGE and Western blotting with an anti-GFP antibody was carried out. Cell extracts indicated by +MG132 were prepared 2 h following the addition of 0.2 mm MG132 and analyzed by Western blot analysis.

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It has previously been proposed that nonstop products may be co-translationally degraded by the proteasome (Ito-Harashima et al. 2007; Dimitrova et al. 2009; Bengtson & Joazeiro 2010). To determine whether peptidyl-tRNA derived from GFP-Rz mRNA is increased in ltn1Δ mutant cells, NuPAGE followed by Western blotting with an anti-GFP antibody was carried out. The level of translated GFP-Rz was increased in ltn1Δ or ltn1Δnot4Δ mutant cells, but peptidyl-tRNA was barely detectable, even in the mutants (Fig. 1D), suggesting that peptidyl-tRNA on stalled 80S ribosomes might not be a substrate for the ligase, as previously proposed (Bengtson & Joazeiro 2010; Brandman et al. 2012). Dom34:Hbs1 is known to be required for the dissociation of ribosomes that are stalled at the 3′ end of GFP-Rz mRNA and for peptide release from tRNA in vivo (Tsuboi et al. 2012). To examine whether peptidyl-tRNA was stabilized in ltn1Δdom34Δ mutant cells, polysome analysis followed by NuPAGE and Western blotting with an anti-GFP antibody was carried out. In ltn1Δ mutant cells, the amount of free peptide in the ribosome-free fractions was increased, but peptidyl-tRNA was barely detectable (Fig. 1E, WT and ltn1Δ panels). The peptidyl-tRNA detected in dom34Δ mutant cells was scarcely increased in ltn1Δdom34Δ mutant cells, although the level of free peptide was drastically increased (Fig. 1E, dom34Δ and ltn1Δdom34Δ panels). The level of free peptide in dom34Δ mutant cells was also increased following treatment with the proteasome inhibitor MG132, but the level of peptidyl-tRNA was not (Fig. 1E, dom34Δ+MG132 panel). These findings indicate that aberrant nonstop polypeptides were drastically stabilized by LTN1 deletion even in the absence of the ribosome dissociation factor Dom34.

Substrate specificity of Ltn1 in protein degradation coupled to translation arrest

Ltn1 destabilizes aberrant products derived from GFP-Rz mRNA lacking a poly(A) tail, indicating that translation arrest induced by the poly(A) sequence is not essential for Ltn1-dependent protein degradation. To address the substrate specificity of Ltn1 for co-translational degradation by the proteasome, the stabilization of the arrest products derived from various reporters shown in Fig. 2A was examined. The arrest products derived from GFP-K12-FLAG-HIS3 (K12) or GFP-rare-FLAG-HIS3 (Rare) mRNA were stabilized in ltn1Δ mutant cells (Fig. 2B, lanes 9–10, 13–14). In contrast, the levels of the full-length products of the K12, Rare and C-K12 reporters were not increased in the ltn1Δ mutant (Fig. 2B, lanes 10, 12, 14, 16, 18). Furthermore, Ltn1 mutants with a deletion or a point mutation in the RING domain were defective in the down-regulation of both arrest proteins (Fig. 2C, lanes 11–12, 15–16). These results indicate that Ltn1 is involved in the proteasomal degradation of translation arrest products, and it is consistent with recent study(Letzring et al. 2013).

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Figure 2. Ltn1 degrades aberrant proteins produced by various types of translation arrest. (A) Schematic drawing of the reporter GGN (GFP-GGN-FLAG-HIS3), K12(AAA) (GFP-K12-FLAG-HIS3) and Rare (GFP-rare codons-FLAG-HIS3) mRNAs. The filled boxes indicate the open reading frames, the lines represent nontranslated regions, and the tract of As denotes the poly (A) tail. The dark box shows the FLAG tag sequence, and X indicates the region of various translation arrest-inducing sequences. (B) Ltn1 destabilizes arrest products produced by RACK1/ASC1-dependent translation arrest. Indicated yeast cells containing the GFP-FLAG-HIS3 (−), GFP-(GGN)12-FLAG-HIS3 (GGN), GFP-K12(AAA)-FLAG-HIS3 (K12), GFP-rare codons-FLAG-HIS3 (Rare) or GFP-FLAG-HIS3-K12 (C-K12) reporter genes were grown in SC-Glu Ura medium, and protein samples were analyzed as in Fig. 1B. When indicated, protein samples were diluted 10-fold. (C) The binding of Ltn1 to E2 enzyme is required for the degradation of arrest products derived from reporter genes. The ltn1Δ mutant cells containing the GFP-FLAG-HIS3 (−), GFP-(GGN)12-FLAG-HIS3 (GGN), GFP-K12(AAA)-FLAG-HIS3 (K12), GFP-rare codons-FLAG-HIS3 (Rare) or GFP-FLAG-HIS3-K12 (C-K12) reporter genes were transformed with plasmids expressing Ltn1 or Ltn1 mutant proteins defective in binding to the E2 enzyme. The levels of arrest products were determined by Western blotting with an anti-GFP antibody. When indicated, protein samples were diluted 10-fold.

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RACK1/ASC1 is required for translation arrest by polybasic amino acid sequences, and the arrest product derived from the GFP-K12-FLAG-HIS3 reporter was not detected in rack1/asc1Δ mutant cells even in the presence of MG132 (Kuroha et al. 2010). Therefore, we examined translation arrest in various reporter genes and found that the levels of the full-length product of the Rare reporter, as well as the K12 and C-K12 reporter, were increased in rack1/asc1Δ mutant cells (Fig. 2B, lanes 11–12, 15–16, 18–19), whereas the levels of the full-length products of GFP-FLAG-HIS3 and GFP-(GGN)12-FLAG-HIS3 reporter mRNAs were not (Fig. 2B, lanes 3–4, 7–8). These data indicate that RACK1/ASC1 is involved in translation arrest induced by poly-lysine sequences and tandem rare codons, but may not GGN repeats sequence. The level of the arrest products of the K12 reporter or the Rare reporter construct was increased in the ltn1Δ mutant (Fig. 2B, lanes 9–10 and 13–14). However, the level of these arrest products in the rack1/asc1Δ mutant was not increased by the deletion of LTN1 (Fig. 2B, lanes 12, 16). These results indicate that Ltn1 is involved in the degradation of arrest products in an RACK1/ASC1-dependent manner.

Synergistic action of two E3 ubiquitin ligases in the proteasomal degradation of arrest products

Two distinct E3 ubiquitin ligases, Not4 and Ltn1, are involved in the degradation of arrest products associated with translation arrest, although the relationship between these two ubiquitin ligases remains largely unknown. To determine the substrate specificity of Not4 and Ltn1, the levels of various translation arrest products were measured in not4Δ and not4Δltn1Δ mutant cells. The levels of arrest products derived from K12 or Rare reporter mRNA were stabilized in both not4Δ and ltn1Δ mutant cells and were dramatically increased in not4Δltn1Δ double mutant cells (Fig. 3A, lanes 9–16). In addition, the Not4 mutant with a point mutation in the RING domain was defective in the down-regulation of arrest products derived from K12 (Fig. 3B). By contrast, the levels of proteins derived from GFP-Rz-FLAG-HIS3 (Rz) or GFP-FLAG-HIS3-NS (NS) mRNA were significantly increased in ltn1Δ or not4Δltn1Δ double mutant cells, but not in not4Δ mutant cells (Fig. 1B, lanes 6–8 and 10–12). These data indicate that Not4 destabilizes the arrest products produced by translation arrest within mRNA, but not those that occur at the 3′ end of mRNA, which is consistent with previous results (Dimitrova et al. 2009).

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Figure 3. Substrate specificity of the two ubiquitin ligases in protein degradation coupled to translation arrest. (A) Wild-type, not4Δ mutant, ltn1Δ mutant and ltn1Δnot4Δ mutant cells containing the indicated reporter genes were grown in SC-Glu Ura medium, and protein samples were analyzed as in Fig. 1B. (B) The binding of Not4 to the E2 enzyme is required for the down-regulation of arrest products. W303not4Δ mutant cells containing GFP-FLAG-HIS3 (−), GFP-K12(AAA)-FLAG-HIS3 (K12) or GFP-rare codons-FLAG-HIS3 (Rare) reporter genes were transformed with plasmids expressing Not4 wild-type (WT) or Not4L35A mutant protein (L35A) defective in binding to the E2 enzyme or control plasmid (−). Protein samples were prepared, and the levels of arrest products were determined by Western blotting with anti-GFP (Top panel) or anti-EF-1α antibodies (Bottom panel). When indicated, protein samples were diluted 10-fold. (C) Schematic drawing of the GFP-K12(AAA)-FLAG-HIS3 and GFP-K12(AAA)-FLAG-HIS3-Rz reporter mRNAs. The filled boxes indicate the open reading frames, the lines represent nontranslated regions, and the tract of As denotes the poly(A) tail. The dark box shows the FLAG tag sequences, and Rz indicates the hammerhead ribozyme sequence that induces self-cleavage. (D) The poly(A) tail is not required for the degradation of arrest products by Not4. Indicated yeast cells containing the GFP-K12(AAA)-FLAG-HIS3 (−) or GFP-K12(AAA)-FLAG-HIS-Rz (-Rz) reporter genes were grown in SC-Glu Ura medium, and protein samples were analyzed as in Fig. 1B. (E) The levels of reporter mRNAs were not affected in the indicated mutants. The relative levels of reporter mRNAs were determined by Northern blotting using DIG-labeled GFP or SCR1 probes, as described previously (Tsuboi et al. 2012).

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Not4 is involved in protein degradation of translation arrest products produced by poly-lysine sequences (Dimitrova et al. 2009), but not those of nonstop proteins(Dimitrova et al. 2009; Bengtson & Joazeiro 2010; Duttler et al. 2013). To address the role of the poly(A) tail in Not4-dependent degradation of arrest products, we examined the degradation of arrest products derived from GFP-K12-FLAG-HIS3-Rz reporter mRNAs lacking a poly(A) tail (Fig. 3C, -Rz constructs). The level of GFP-K12-FLAG-HIS3-Rz mRNA was lower than that of GFP-K12-FLAG-HIS3 mRNA in wild-type or not4Δltn1Δ mutant cells (Fig. 3E, lanes 1–2 and 7–8), and there was no significant difference between the levels of these mRNAs in not4Δ or ltn1Δ single mutants (Fig. 3E, lanes 3–4 and 5–6). In contrast, the level of full-length product derived from GFP-K12-FLAG-HIS3-Rz was also significantly decreased (Fig. 3D, even lanes), and the levels of arrest products were significantly increased following LTN1 deletion in the NOT4 (Fig. 3D, lanes 1–2 and 5–6) or not4Δ mutant background (Fig. 3D, lanes 3–4 and 7–8). In addition, the levels of arrest products were slightly increased as a result of NOT4 deletion in the LTN1 (Fig. 3D, lanes 1–4) or ltn1Δ mutant background (Fig. 3D, lanes 5–8). But the levels of arrest products were not affected by artificial removal of the poly(A) tail in any cases; these data suggest that the poly(A) tail may not be required for the degradation of arrest products by Ltn1 and Not4.

Ubiquitin-dependent degradation may occur after peptide release from the tRNA

Cdc48 is a member of the hexameric AAA family of proteins that possess two ATPase domains, and this protein converts energy from ATP hydrolysis to structurally remodel or unfold ubiquitinated proteins for efficient degradation by the proteasome (Bebeacua et al. 2012). Cdc48 is also involved in the ER-associated degradation (ERAD) pathway, which is the most thoroughly characterized protein quality control system (Meyer 2012). The current model for ERAD proposes that aberrant proteins are translocated to the cytosol from the ER, and then an Ufd1-Npl4-Cdc48 complex extracts the aberrant protein for degradation by the proteasome (Brandman et al. 2012; Defenouillere et al. 2013; Verma et al. 2013). Cdc48 associates with the 60S subunit as a complex with Ltn1 and other novel factors required for the degradation of arrest products produced by translation arrest mediated by poly-lysine sequences (Brandman et al. 2012; Defenouillere et al. 2013). To address the role of Cdc48 in the degradation of aberrant nonstop proteins and the degradation of arrest products, the expression of the K12 and Rare reporter proteins was examined in cdc48-3 mutant cells in which the degradation of ERAD substrates was defective at the permissive temperature. Western blotting with an anti-GFP antibody was used to detect putative degradation intermediates of translation arrest products derived from GFP-FLAG-HIS3-C-K12 (C-K12) reporter mRNA (Fig. 4A, lane 18), but not of other reporter proteins in the ltn1Δ mutant (Fig. 4A, lanes 1–16). We found that cdc48-3 mutant cells always express less reporter proteins than the wild-type cells, suggesting that this may be due to overall reduction in translation activity in the mutant. To confirm that degradation intermediates stabilized in cdc48-3 mutant cells containing an intact GFP moiety, a 3xHA tag sequence was inserted at the amino terminus of the reporter genes (Fig. 4B). The stabilized products derived from 3HA-GFP-FLAG-HIS3-C-K12 (HA-C-K12) and 3HA-GFP-FLAG-HIS3-Rare (HA-C-Rare) mRNA were detected using anti-HA antibodies (Fig. 4C, top panel), and these findings indicated that the stabilized products derived from the C-K12 reporter mRNA contained intact GFP and part of the His3 protein. Although the level of intermediate product derived from the C-K12 reporter mRNA was significantly reduced in the ltn1Δcdc48-3 mutant, the level of the full-length C-K12 product was increased (Fig. 4A, lanes 19-20), and the arrest products derived from K12 or Rare reporter genes were also detected (Fig. 4A, lanes 11–12, 15–16). We also carried out Western blot with anti-FLAG antibodies to show the presence of the FLAG, which would indicate that the GFP is intact in these intermediate products (Fig. 4C, middle panel). These results indicate that Ltn1 acts upstream of Cdc48, which is consistent with the hypothesis that, in the degradation of arrest products, Ltn1 may ubiquitinate peptidyl-tRNA on the 60S ribosome and that the proteasome degrades aberrant polypeptides from the carboxyl terminus after release from the tRNA. These results also suggest that Cdc48 may be required for unfolding of the GFP moiety of the aberrant protein for complete degradation by the proteasome.

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Figure 4. Ltn1-dependent degradation of aberrant proteins is triggered from the carboxyl terminus. (A) Wild-type, cdc48-3, ltn1Δ and ltn1Δcdc48-3 cells were transformed with the indicated reporter genes and grown in SC-Glu Ura medium at the permissive temperature of 25 °C. Protein samples were analyzed as in Fig. 1B. When indicated, protein samples were diluted 10-fold. (B) Schematic drawing of the C-K12 (3HA-GFP-FLAG-HIS3-K12(AAA)) and C-Rare (3HA-GFP-FLAG-HIS3-rare codons) reporter mRNAs for detecting the degradation intermediates stabilized in the cdc48-3 temperature-sensitive mutant. The filled boxes indicate the open reading frames, the lines represent nontranslated regions, and the tract of As denotes the poly(A) tail. Two dark boxes show the HA and FLAG tag sequences, and X indicates the translation arrest-inducing sequences containing K12 or Rare. (C) The degradation intermediates stabilized in the cdc48 temperature-sensitive mutant contained the entire GFP region. Wild-type and cdc48-3 mutant cells were transformed with p416GPDp-3HA-GFP-FLAG-HIS3-K12(AAA) (HA-C-K12) or p416GPDp-3HA-GFP-FLAG-HIS3-rare codons (HA-C-Rare). Cells were grown in SC-Glu Ura medium at 25 °C, and protein samples were analyzed by Western blotting with anti-HA (top panel) anti-FLAG (middle panel) or anti-aeEF-1α (bottom panel) antibodies.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Previous studies have showed that mRNA quality control systems stimulate the degradation of aberrant mRNA to prevent the potentially harmful products derived from aberrant mRNAs. Recent studies on quality control systems induced by abnormal translation elongation and termination have revealed that both aberrant mRNAs and proteins are subjected to rapid degradation (Ito-Harashima et al. 2007; Dimitrova et al. 2009; Bengtson & Joazeiro 2010; Kuroha et al. 2010; Brandman et al. 2012; Defenouillere et al. 2013; Shao et al. 2013; Verma et al. 2013). In this study, we examined the mechanism of co-translational degradation of aberrant proteins by the proteasome, as well as the roles of two E3 ubiquitin ligases, ribosome-stalling factor RACK1/ASC1 and the stalled ribosome dissociation factor Dom34:Hbs1. Our results strongly suggest that Ltn1 and Not4 have synergistic effects on the degradation of arrest products (Fig. 3) and that the poly(A) tail may not be required for the degradation of arrest products by these two ligases (Figs 1, 3). Ltn1 plays a crucial role in the degradation of arrest products derived from mRNAs containing arrest-inducing sequences, including polybasic amino acid sequences and rare codons (Fig. 2). Furthermore, Ltn1 may recognize peptidyl-tRNA on stalled ribosomes and ubiquitinate this complex for degradation by the proteasome. In contrast, Not4 may bind to the 80S ribosome that is stalled within the mRNA, but not at the 3′ end of the mRNA, which is consistent with the ribosome binding specificity of these ligases, as Not4 is found in the polysome fractions (Dimitrova et al. 2009) and Ltn1 is mainly distributed in the 60S subunit (Bengtson & Joazeiro 2010; Brandman et al. 2012). We did not directly detect ubiquitination of aberrant proteins on polysomes. Therefore, whether Ltn1 ubiquitinates polypeptides from aberrant mRNAs before or after peptidyl-tRNA hydrolysis is still unknown. Based on these results, we have proposed models for the degradation of arrest products. When a ribosome is stalled within mRNA during translation elongation in a RACK1/ASC1-dependent manner, it is dissociated, and Ltn1 may ubiquitinate the peptidyl-tRNA on the 60S subunit or the peptide released from tRNA. Moreover, Ltn1 and Not4 have synergistic effects on the degradation of arrest products, and Not4 may bind to the stalled 80S ribosome, but not to the 60S subunit containing the peptidyl-tRNA.

Translation arrest due to poly-arginine sequences or tandem rare codons was defective in the RACK1/ASC1 mutant (Fig. 2) (Kuroha et al. 2010). Moreover, the level of the protein products derived from the reporter gene containing poly-lysine sequences within the ORF was drastically increased in the RACK1/ASC1 deletion mutant, whereas the levels of arrest products were significantly decreased in the RACK1/ASC1 deletion mutant even in the absence of Ltn1 (Fig. 2). The precise mechanism by which RACK1/ASC1 induces translation arrest by two different cis-elements, including specific nascent peptide sequences and a low abundance of aminoacyl-tRNA, has largely remained unknown. However, 40S-bound RACK1/ASC1 is important for regulating translation by sensing aberrant elongation reactions. In addition, RACK1/ASC1 makes extensive contacts with the phosphate backbone and bases of h39 and h40 of 18S rRNA (Rabl et al. 2011). RACK1/ASC1 is also associated with rpS16e, S17e and S3e over a large contact surface (Rabl et al. 2011), and these ribosome proteins near the exit tunnel of the 40S subunit play crucial roles in the fidelity of translation elongation. Therefore, translation defects in RACK1/ASC1 mutants must be investigated to understand the precise function of RACK1/ASC1 in translation arrest induced by specific cis-elements.

Dom34:Hbs1 stimulates the decay of nonstop mRNAs and 5′ NGD intermediates by dissociating ribosomes that are stalled at the 3′ end of the mRNA and plays an important role in NSD and NGD (Tsuboi et al. 2012). The results in this study showed that both Not4 and Ltn1 are involved in the degradation of arrest products associated with RACK1/ASC1-dependent translation arrest, whereas the Dom34:Hbs1 complex is only minimally involved in the rapid degradation of arrest products. When a ribosome is stalled within an mRNA during translation elongation, it may be dissociated in a Dom34:Hbs1-independent manner. Subsequently, the peptidyl-tRNA on the 60S subunit is ubiquitinated by Ltn1, leading to degradation by the proteasome. In contrast to ribosome stalling within the mRNA, when the ribosome is stalled at the 3′ end of aberrant mRNAs lacking a termination codon, there are two potential consequences (Fig. S2 in Supporting Information). First, the Dom34:Hbs1 complex may bind to an empty A-site and stimulate dissociation of the ribosome, leading to the rapid degradation of aberrant mRNAs, as previously reported (Tsuboi et al. 2012). The GTPase activity of Hbs1 is stimulated by ribosome and induces the release of Hbs1 from Dom34/Pelota that is required for the split of ribosome by main ribosome-recycling factor Rli1/ABCE1(Pisarev et al. 2010; Shoemaker et al. 2010; Pisareva et al. 2011; Shoemaker & Green 2011; Becker et al. 2012). Rli1/ABCE1 interacts with carboxyl-terminal domain of Dom34/Pelota and splits 80S ribosome into subunits in an ATP hydrolysis-dependent manner in vitro (Pisareva et al. 2011; Shoemaker & Green 2011; Becker et al. 2012). During subunit dissociation by the Dom34:Hbs1 complex, the polypeptide may be released from the tRNA, but not subjected to ubiquitination by Ltn1. In the alternative pathway, the stalled ribosome may be dissociated into the 40S subunit and the 60S subunit containing the peptidyl-tRNA. Then, the peptidyl-tRNA on the 60S subunit may be subjected to Ltn1-dependent ubiquitination and degraded by the proteasome. The protein quality control system for nonstop products produced from nonstop mRNA containing a poly(A) tail differs from that associated with the degradation of aberrant nonstop proteins produced from stop codon-less mRNA lacking a poly(A) tail. Translation of a poly(A) tail induces strong translation arrest (Inada & Aiba 2005; Ito-Harashima et al. 2007). However, some populations of ribosomes may be stalled at the 3′ end of mRNA, dissociated by Dom34:Hbs1 and degraded by the exosome. Moreover, neither Not4 nor RACK1/ASC1 is essential for the degradation of nonstop proteins derived from nonstop mRNA (Dimitrova et al. 2009; Kuroha et al. 2010). We did not directly detect ubiquitination of aberrant proteins derived from GFP-Rz mRNA on polysomes (Fig. 1E), and there are two possibilities that Ltn1 may ubiquitinate peptidyl-tRNAs and/or peptides released from the peptidyl-tRNAs derived from nonstop mRNA lacking a termination codon.

Cdc48/p97 promotes the degradation of aberrant nascent polypeptides bound to the ribosome (Brandman et al. 2012; Verma et al. 2013). It has been showed that the ubiquitinated proteins were accumulated and associated with ribosomes, and cdc48-3 cells accumulated even peptidyl-tRNAs of the nonstop mRNA with poly(A). This seems to be inconsistent with the fact that Ltn1, acting upstream of Cdc48, accumulate polypeptides but not peptidyl-tRNAs from nonstop mRNAs without poly(A) in Fig. 1C. One possibility is that this is due to the condition of the harvest. In Verma et al.'s study, cdc48-3 ts cells were harvested after the 2-h growth at restricted temperature. In contrast, we have harvested cdc48-3 cells grown at permissive temperature and might show the less severe defects than that at restricted temperature. The other possibility is that Ltn1 acts on peptidyl-tRNAs and polypeptides derived from aberrant mRNAs differently according to the existence of poly(A) on the mRNAs. Indeed, Cdc48 is also required for efficient degradation of a approximately 30 kDa intermediate without the C-terminal region of C-K12, whose mRNA is not only an arrested mRNA but also a mimic of a non-stop mRNA with poly(A). These suggest that Cdc48 may have several action points in degradation of translation products from arrested mRNA. Although the mechanism by which E3 ubiquitin ligases recognize stalled ribosomes remains to be resolved, it is clear that nascent peptides are subjected to different protein quality control systems and that improper translation elongation is sufficient to induce degradation. Our results suggest that Cdc48 may unfold aberrant proteins for efficient proteasome-dependent degradation in the degradation of arrest products and aberrant nonstop proteins. We propose that Cdc48 may play two roles in protein quality control systems coupled to aberrant translation: dissociation of the peptidyl-tRNA on the 60S subunit and unfolding of aberrant proteins derived from aberrant mRNAs. Further experiments are needed to make clear the function of Cdc48 in the degradation of arrest products derived from various aberrant mRNAs.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Strains and plasmids

The yeast strains and plasmids used in this study are listed in Table S1 (Supporting Information), and the oligonucleotides used for plasmid construction are described in Table S2 (Supporting Information).

Detection of peptidyl-tRNA

Peptidyl-tRNA was detected using cell extracts and polysome fractions using NuPAGE followed by Western blotting as described previously (Tsuboi et al. 2012). To destroy the RNA moiety of peptidyl-tRNA, RNase A was added to the samples at a final concentration of 10 μg/ml and was incubated at 37 °C for 10 min.

Polysome analysis

Yeast cells were grown exponentially at 30 °C and harvested by centrifugation. Cell extracts were prepared as described previously (Inada & Aiba 2005). The equivalent of 50 A260 units was then layered onto linear 10%–50% sucrose density gradients. Sucrose gradients (10%–50% sucrose in 10 mm Tris-acetate pH 7.4, 70 mm ammonium acetate, 4 mm magnesium acetate) were prepared in 25 × 89 mm polyallomer tubes (Beckman Coulter) using a gradient master. Crude extracts were layered on top of the sucrose gradients and then centrifuged at 150 000 g in a P28S rotor (Hitachi Koki, Japan) for 2.5 h at 4 °C. Gradients were then fractionated (TOWA lab, Tsukuba), and polysome profiles were generated by continuous absorbance measurements at 254 nm using a single path UV-1 optical unit (ATTO Biomini UV-monitor) connected to a chart recorder (ATTO, digital mini-recorder). Fractions of equal volume were collected and processed for NuPAGE followed by Western blotting, as described above.

Plasmid construction

Plasmids expressing various Ltn1 proteins were constructed as follows. To construct pIT2125 (p415GPDp-HA-FLAG), a SpeI-XhoI fragment of HA-LTN1 was amplified by PCR using the two primers OKK228 and OKK229, and this product was then inserted into the SpeI-XhoI sites of p415ADHp. To construct pIT2126 (p415GPDp-HA-LTN1ΔRING) or pIT2127 (p415GPDp-HA-LTN1-W1542E), mutations were introduced by site-directed mutagenesis using the primers listed in Table S2 (Supporting Information). To construct pIT2128 (p415GPDp-LTN1-FLAG), pIT2129 (p415GPDp-LTN1ΔRING-FLAG) or pIT2130 (p415GPDp-LTN1-W1542E-FLAG), SpeI-XhoI fragments of pIT2125 (p415GPDp-HA-FLAG), pIT2126 (p415GPDp-HA-LTN1ΔRING) or pIT2127 (p415GPDp-HA-LTN1-W1542E) were amplified by PCR using the two primers OIT1802 and OIT1884, and the product was then inserted into the SpeI-XhoI sites of p415ADHp.

To construct the pIT2131 (p416GPD-GFP-K12(AAA)-FLAG-HIS3-Rz) reporter, the hammerhead ribozyme sequence (Rz) was inserted into pIT2051 (pGPDp-GFP-K12(AAA)-FLAG-HIS3) by site-directed mutagenesis using the primers listed in Table S2 (Supporting Information).

To construct pIT2132 (p416GPD-GFP(BHΔ)-FLAG-HIS3), mutations to disrupt the BamHI site were introduced into pSA144 (pGPDp-GFP-FLAG-HIS3-CYC1ter) by site-directed mutagenesis using the primers listed in Table S2 (Supporting Information). The two oligonucleotides OKK80 and OKK81 were annealed and inserted into the XbaI site of pIT2132 to create the pIT2133 (p416GPD-3HA-GFP(BHΔ)-FLAG-HIS3) reporter. The two oligonucleotides OIT980 and OIT981 were annealed and inserted into the SpeI site of pIT2133 to create pIT2134 (p416GPD-3HA-GFP(BHΔ)-K12(AAA)-FLAG-HIS3). The two oligonucleotides OIT431 and OIT432 were annealed and inserted into the SpeI site of pIT2133 to create pIT2135 (p416GPD-3HA-GFP(BHΔ)-rare-FLAG-HIS3). The two oligonucleotides OIT1181 and OIT1182 were annealed and inserted into the SpeI site of pIT2133 to create pIT2136 (p416GPD-3HA-GFP(BHΔ)-Rz-FLAG-HIS3). The two oligonucleotides OIT2147 and OIT2148 were annealed and inserted into the BamHI site of pIT2133 to create pIT2137 (p416GPD-3HA-GFP(BHΔ)-FLAG-HIS3-K12(AAA)). The two oligonucleotides OIT2145 and OIT2146 were annealed and inserted into the BamHI site of pIT2134 to create pIT2138 (p416GPD-3HA-GFP(BHΔ)-FLAG-HIS3-rare).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors thank Dr Kunio Nakatsukasa and Dr Takumi Kamura for the yeast strains, plasmids and valuable discussion and comments. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas of ‘RNA regulation’ (No. 20112006) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T.I). The authors appreciate constructive discussion with Dr Onn Brandman and Dr Jonathan Weissman.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Bai, Y., Salvadore, C., Chiang, Y.C., Collart, M.A., Liu, H.Y. & Denis, C.L. (1999) The CCR4 and CAF1 proteins of the CCR4-NOT complex are physically and functionally separated from NOT2, NOT4, and NOT5. Mol. Cell. Biol. 19, 66426651.
  • Bebeacua, C., Forster, A., McKeown, C., Meyer, H.H., Zhang, X. & Freemont, P.S. (2012) Distinct conformations of the protein complex p97-Ufd1-Npl4 revealed by electron cryomicroscopy. Proc. Natl Acad. Sci. USA 109, 10981103.
  • Becker, T., Franckenberg, S., Wickles, S., Shoemaker, C.J., Anger, A.M., Armache, J.P., Sieber, H., Ungewickell, C., Berninghausen, O., Daberkow, I., Karcher, A., Thomm, M., Hopfner, K.P., Green, R. & Beckmann, R. (2012) Structural basis of highly conserved ribosome recycling in eukaryotes and archaea. Nature 482, 501506.
  • Bengtson, M.H. & Joazeiro, C.A. (2010) Role of a ribosome-associated E3 ubiquitin ligase in protein quality control. Nature 467, 470473.
  • Brandman, O., Stewart-Ornstein, J., Wong, D., Larson, A., Williams, C.C., Li, G.W., Zhou, S., King, D., Shen, P.S., Weibezahn, J., Dunn, J.G., Rouskin, S., Inada, T., Frost, A. & Weissman, J.S. (2012) A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress. Cell 151, 10421054.
  • Chen, L., Muhlrad, D., Hauryliuk, V., Cheng, Z., Lim, M.K., Shyp, V., Parker, R. & Song, H. (2010) Structure of the Dom34-Hbs1 complex and implications for no-go decay. Nat. Struct. Mol. Biol. 17, 12331240.
  • Defenouillere, Q., Yao, Y., Mouaikel, J., Namane, A., Galopier, A., Decourty, L., Doyen, A., Malabat, C., Saveanu, C., Jacquier, A. & Fromont-Racine, M. (2013) Cdc48-associated complex bound to 60S particles is required for the clearance of aberrant translation products. Proc. Natl Acad. Sci. USA 110, 50465051.
  • Dimitrova, L.N., Kuroha, K., Tatematsu, T. & Inada, T. (2009) Nascent peptide-dependent translation arrest leads to Not4p-mediated protein degradation by the proteasome. J. Biol. Chem. 284, 1034310352.
  • Doma, M.K. & Parker, R. (2006) Endonucleolytic cleavage of eukaryotic mRNAs with stalls in translation elongation. Nature 440, 561564.
  • Duttler, S., Pechmann, S. & Frydman, J. (2013) Principles of cotranslational ubiquitination and quality control at the ribosome. Mol. Cell 50, 379393.
  • van den Elzen, A.M., Henri, J., Lazar, N., Gas, M.E., Durand, D., Lacroute, F., Nicaise, M., van Tilbeurgh, H., Seraphin, B. & Graille, M. (2010) Dissection of Dom34-Hbs1 reveals independent functions in two RNA quality control pathways. Nat. Struct. Mol. Biol. 17, 14461452.
  • Frischmeyer, P.A., van Hoof, A., O'Donnell, K., Guerrerio, A.L., Parker, R. & Dietz, H.C. (2002) An mRNA surveillance mechanism that eliminates transcripts lacking termination codons. Science 295, 22582261.
  • Gandhi, R., Manzoor, M. & Hudak, K.A. (2008) Depurination of Brome mosaic virus RNA3 in vivo results in translation-dependent accelerated degradation of the viral RNA. J. Biol. Chem. 283, 3221832228.
  • van Hoof, A. & Parker, R. (2002) Messenger RNA degradation: beginning at the end. Curr. Biol. 12, R285R287.
  • Inada, T. & Aiba, H. (2005) Translation of aberrant mRNAs lacking a termination codon or with a shortened 3′-UTR is repressed after initiation in yeast. EMBO J. 24, 15841595.
  • Ito-Harashima, S., Kuroha, K., Tatematsu, T. & Inada, T. (2007) Translation of the poly(A) tail plays crucial roles in nonstop mRNA surveillance via translation repression and protein destabilization by proteasome in yeast. Genes Dev. 21, 519524.
  • Izawa, T., Tsuboi, T., Kuroha, K., Inada, T., Nishikawa, S. & Endo, T. (2012) Roles of dom34:Hbs1 in nonstop protein clearance from translocators for normal organelle protein influx. Cell Rep. 2, 447453.
  • Kobayashi, K., Kikuno, I., Kuroha, K., Saito, K., Ito, K., Ishitani, R., Inada, T. & Nureki, O. (2010) Structural basis for mRNA surveillance by archaeal Pelota and GTP-bound EF1alpha complex. Proc. Natl Acad. Sci. USA 107, 1757517579.
  • Kuroha, K., Akamatsu, M., Dimitrova, L., Ito, T., Kato, Y., Shirahige, K. & Inada, T. (2010) Receptor for activated C kinase 1 stimulates nascent polypeptide-dependent translation arrest. EMBO Rep. 11, 956961.
  • Letzring, D.P., Wolf, A.S., Brule, C.E. & Grayhack, E.J. (2013) Translation of CGA codon repeats in yeast involves quality control components and ribosomal protein L1. RNA 19, 12081217.
  • Meyer, H. (2012) p97 complexes as signal integration hubs. BMC Biol. 10, 48.
  • Mulder, K.W., Inagaki, A., Cameroni, E., Mousson, F., Winkler, G.S., De Virgilio, C., Collart, M.A. & Timmers, H.T. (2007) Modulation of Ubc4p/Ubc5p-mediated stress responses by the RING-finger-dependent ubiquitin-protein ligase Not4p in Saccharomyces cerevisiae. Genetics 176, 181192.
  • Panasenko, O., Landrieux, E., Feuermann, M., Finka, A., Paquet, N. & Collart, M.A. (2006) The yeast Ccr4-Not complex controls ubiquitination of the nascent-associated polypeptide (NAC-EGD) complex. J. Biol. Chem. 281, 3138931398.
  • Panasenko, O.O. & Collart, M.A. (2012) Presence of Not5 and ubiquitinated Rps7A in polysome fractions depends upon the Not4 E3 ligase. Mol. Microbiol. 83, 640653.
  • Pisarev, A.V., Skabkin, M.A., Pisareva, V.P., Skabkina, O.V., Rakotondrafara, A.M., Hentze, M.W., Hellen, C.U. & Pestova, T.V. (2010) The role of ABCE1 in eukaryotic posttermination ribosomal recycling. Mol. Cell 37, 196210.
  • Pisareva, V.P., Skabkin, M.A., Hellen, C.U., Pestova, T.V. & Pisarev, A.V. (2011) Dissociation by Pelota, Hbs1 and ABCE1 of mammalian vacant 80S ribosomes and stalled elongation complexes. EMBO J. 30, 18041817.
  • Rabl, J., Leibundgut, M., Ataide, S.F., Haag, A. & Ban, N. (2011) Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1. Science 331, 730736.
  • Shao, S., von der Malsburg, K. & Hegde, R.S. (2013) Listerin-dependent nascent protein ubiquitination relies on ribosome subunit dissociation. Mol. Cell 50, 637648.
  • Shoemaker, C.J., Eyler, D.E. & Green, R. (2010) Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay. Science 330, 369372.
  • Shoemaker, C.J. & Green, R. (2011) Kinetic analysis reveals the ordered coupling of translation termination and ribosome recycling in yeast. Proc. Natl Acad. Sci. USA 108, E1392E1398.
  • Tsuboi, T., Kuroha, K., Kudo, K., Makino, S., Inoue, E., Kashima, I. & Inada, T. (2012) Dom34:Hbs1 plays a general role in quality-control systems by dissociation of a stalled ribosome at the 3′ end of aberrant mRNA. Mol. Cell 46, 518529.
  • Verma, R., Oania, R.S., Kolawa, N.J. & Deshaies, R.J. (2013) Cdc48/p97 promotes degradation of aberrant nascent polypeptides bound to the ribosome. ELife 2, e00308.
  • Wilson, M.A., Meaux, S. & van Hoof, A. (2007) A genomic screen in yeast reveals novel aspects of nonstop mRNA metabolism. Genetics 177, 773784.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
gtc12106-sup-0001-FigureS1-S2-TableS1-S2.docWord document365K

Figure S1 The levels of Ltn1 and Not4 mutant proteins.

Figure S2 Protein quality control systems associated with nonstop mRNA surveillance in yeast.

Table S1 Yeast strains and plasmids used in this study

Table S2 List of oligonucleotides primers used for plasmid construction

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