![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Unité de Biochimie Cellulaire, UMR 7098 CNRS-Université Paris 6, 9 quai Saint-Bernard, 75005 Paris, France,1 Université de Rennes 1, CNRS UMR 6061, IFR 140 GFAS, 2 Avenue Pr. Léon Bernard, 35043 Rennes Cedex, France,2 Faculté de Médecine et de Pharmacie, Université Cheick Anta Diop, Dakar, Sénégal3
Received 2 March 2005/ Returned for modification 25 March 2005/ Accepted 14 April 2005
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The C-terminal regions of eRF3 proteins are highly conserved through evolution and carry the four canonical GTP-binding motifs of the GTPase superfamily (2). This domain is essential for translation termination and interaction with eRF1. The N-terminal region varies in both length and sequence among species. In yeast, it is neither essential for cell viability nor required for termination but is responsible for prion-like [PSI+] factor formation (29, 31). Otherwise, this domain participates in the interaction with eRF1 (15, 30) and is involved in eRF3 binding to the poly(A)-binding protein, PABP (14). In vivo, eRF3 interacts simultaneously with eRF1 and PABP when the latter is bound to the translation initiation factor eIF-4F (37). This complex mediates ribosome recycling and ensures the coupling between termination and initiation of translation. Moreover, in yeast, eRF3-PABP interaction couples translation to mRNA decay (17). Recently, the crystal structure of Schizosaccharomyces pombe N-terminally truncated eRF3 revealed a strong overall similarity with the elongation factors EF-Tu and eEF-1A, but also local structural changes that affect nucleotide and Mg2+ binding to eRF3 (23). Indeed, eRF3 has negligible affinity for GDP at physiological Mg2+ concentration, implying that the GDP-to-GTP transition of eRF3 would not require a guanine exchange factor. In addition, the interaction domain with eRF1 was localized close to the eRF3 C terminus, and it was shown that eRF3 N-terminal extension can block this domain, potentially regulating the interaction between the two factors (23).
Two distinct genes encoding eRF3 were identified in the human, mouse, and rat genomes, but not in the recently available chicken genome. These genes, called GSPT1/eRF3a and GSPT2/eRF3b (15, 16, 19), are located on human chromosomes 16 and X, respectively. The eRF3a gene contains several intronic sequences, whereas the eRF3b gene has none. At the mRNA level, eRF3a and eRF3b differ in tissue distribution and in expression during cell cycle progression (15, 16). eRF3b mRNA is poorly expressed in most mouse tissues tested except the brain, whereas eRF3a mRNA is abundant in all tissues, and its level varies during the cell cycle. The encoded proteins, eRF3a and eRF3b, share 87% identity, most of the differences being concentrated in their N-terminal domains. Both proteins bind to eRF1 and stimulate eRF1 release activity in vitro, suggesting that both can play a role in translation termination (15, 19). However, in yeast, the interaction of mouse eRF3a with eRF1 in the two-hybrid system is much less efficient than that of mouse eRF3b (15), and mouse eRF3b, but not eRF3a, can substitute for yeast eRF3 (25). Moreover, in human cells, the overexpression of eRF3b, but not that of eRF3a, has an antisuppressor effect (19, 26). These results call into question the role of eRF3a, and finally, whether both proteins can function as translation termination factors in vivo has not been clearly determined.
In the present work, we first analyzed the distributions of the eRF3a and eRF3b proteins in various human cell lines and mouse brain, and second, we investigated the roles of eRF3a and eRF3b in translation termination. For this purpose, we silenced the expression of each gene in cultured human cells, using short interfering RNAs (siRNAs). The effect of silencing on translation termination efficiency was examined by measuring the readthrough at a premature nonsense codon in a reporter mRNA. Our results show that eRF3a is present in all cell lines tested, whereas eRF3b is found only in mouse brain. In human 293 cells, we found that eRF3a silencing induces a significant increase in readthrough, whereas eRF3b silencing has no detectable effect. We also show that both eRF3a and eRF3b overexpression alleviates the effect of eRF3a silencing on readthrough efficiency. In addition, eRF3a depletion reduces the intracellular level of eRF1 protein by affecting its stability. Thus, in contrast with the literature on eRF3a and eRF3b, these results suggest that eRF3a is the major factor acting in translation termination in mammalian cells and that its expression level influences eRF1 protein stability, regulating the formation of the translation termination complex. We also demonstrate that eRF3b, which seems to be poorly expressed in mammalian cells, can substitute for eRF3a in the translation termination process.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Antibodies.
The antibodies against human eRF3a, eRF3b, and eRF1; mouse eRF3a and eRF3b; and yeast eRF3 were produced by Eurogentec (Belgium). Rabbits were immunized with synthetic peptides derived from the protein sequence. Antibodies were then obtained by affinity purification of the antisera using the corresponding immunogenic peptide: peptide GGRAAPVESSQEE of human eRF3a, peptide GKRMGRGAPVEPSRE of human eRF3b (Fig. 1A), a mixture of peptides VQQRLKLYNKVPPNG and EYQGGDDEFFDLDDY of human eRF1, peptide GGSSGDGRPPEESTQ of mouse eRF3a (mGSPT1), peptide SAPSGDGIAPAAMA of mouse eRF3b (mGSPT2), and a mixture of peptides MSDSNQGNNQQNYQ and LEKGTNRKSKKPPA of S. cerevisiae eRF3. The antibodies directed against Xenopus laevis eRF3 (XRF3) were described previously (26). The anti-
-tubulin (DM1A), anti-rabbit immunoglobulin G (IgG), and anti-mouse IgG peroxidase-linked antibodies were from Amersham Biosciences (England). The anti-Neo and anti ß-galactosidase rabbit antibodies were from 5prime-3prime (France).
|
|
Transfection was carried out by the calcium phosphate coprecipitation method with 20 µg of DNA per 100-mm-diameter cell plate at 50% confluency. After overnight incubation, the medium and precipitate were removed and replaced with 10 ml of fresh medium. Following a further 24-h incubation, cells were collected by scraping them in 10 ml of PBS and pelleted.
To establish the 559C stable cell line, 1 µg of pCMV-nls559C was transfected in 293 cells. After overnight incubation, the cells were replated by diluting them 1:10 in complete DMEM. Clones were selected for 2 weeks in complete DMEM containing 800 µg/ml G418 (Roche) and screened for ß-galactosidase activity by transfection of plasmid ptRNAam expressing a human UAG suppressor tRNA (26) and X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) staining. Five positive clones, 559C-1 to 559C-5, were selected and stored frozen in liquid nitrogen. Subsequently, 559C stable cell lines were propagated in the absence of G418 selection for about 30 passages without observable loss of ß-galactosidase activity.
RNA isolation and Northern blot analysis. Total RNA was extracted as previously described (6). For Northern blot analysis, 20 µg of total RNA was subjected to electrophoresis in a 1% agarose-formaldehyde gel, blotted, and UV cross-linked onto a Hybond-N+ membrane (Amersham Biosciences). The membrane was sequentially hybridized in CHURCH solution (7) with randomly 32P-labeled fragments of human eRF3a (GSPT1) cDNA, human eRF1 cDNA, the lacZ gene of plasmid pCMV-nls559C, and human ß-actin cDNA. Following hybridization, the membrane was washed under stringent conditions, exposed to X-ray film, stripped, and reprobed under the same conditions.
Western blot analysis. Cell pellets were resuspended in 100 µl of PBS containing a 2x Complete EDTA-free cocktail of protease inhibitors (Roche), 1 µg/ml pepstatin, and 10 mM EDTA. The cells were lysed by sonication on ice and centrifuged for 5 min at 16,000 x g, and the supernatant was retained as cell extract. Mouse brain extracts were obtained as previously described (24). The protein concentrations of extracts were determined using the Micro BCA Protein Assay Reagent Kit (Pierce), with bovine serum albumin used as a standard. For each sample, 20 µg of total protein was loaded on an 8% polyacrylamide gel and subjected to electrophoresis. Proteins were subsequently electrotransfered onto a Hybond-C Extra membrane (Amersham Biosciences), and the membrane was blocked for 1 h in Tris-buffered saline (TBS)-Tween solution (20 mM Tris-HCl, pH 7.6, 140 mM NaCl, 0.2% Tween 20) containing 2% milk. The membrane was incubated overnight with primary antibodies at the appropriate dilution in TBS-Tween, washed five times for 10 min in TBS-Tween, and probed for 45 min with anti-rabbit IgG peroxidase-linked secondary antibodies at 1:30,000 dilution in TBS-Tween. The membrane was washed again five times in TBS-Tween and visualized by chemiluminescence and exposure to X-ray film.
Readthrough assay. The pellets of electroporated cells were resuspended in 300 µl of 100 mM sodium phosphate buffer, pH 8. The cells were lysed by four cycles of freezing and thawing, and the cell lysates were centrifuged at 12,000 x g for 30 min at 4°C. The resulting supernatants were assayed for total proteins and ß-galactosidase activity. For each sample, the total protein concentration was determined using the Micro BCA Protein Assay Reagent Kit (Pierce). The ß-galactosidase assays were performed using the Luminescent Beta-galactosidase Detection Kit II (Clontech). Following the manufacturer's instuctions, ß-galactosidase reactions were carried out in duplicate with 15 µl of extract and 200 µl of reaction buffer containing Galacton-Star substrate. After 1 h at room temperature, the ß-galactosidase activity was measured as relative light units using a single-photon-counting program on a scintillation counter. The ß-galactosidase activity was expressed as relative light units/µg of total protein, and the readthrough efficiencies were calculated as sample activity relative to negative control activity (negative controls are given in the figure legends).
Pulse-chase labeling of cell proteins and immunoprecipitation.
Three days after electroporation, cells were preincubated for 1 h in culture medium lacking methionine and cysteine and metabolically labeled for 2 h with 0.3 mCi of PRO-MIX L-[35S] in vitro Cell Labeling Mix (Amersham Biosciences) in 5 ml of culture medium lacking methionine and cysteine. The medium was removed, and the cell monolayer was washed with PBS and chased with complete DMEM. At various times postchase, cells were collected by scraping them in 10 ml of PBS and pelleted. The cells were lysed by sonication, and the extracts used for immunoprecipitation were prepared as described above for Western blot analysis. Immunoprecipitation was performed as described previously (39) with 2 x 107 cpm of radiolabeled cell extract and either 5 µl of anti-eRF1 antibodies or 2.5 µl of anti--tubulin antibodies. Immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gels were fixed in 30% ethanol-10% acetic acid, dried, and developed by autoradiography. The protein bands of interest were quantified using the BAS1000 Fuji image plate program version 2.0.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
6-fold and 3-fold, respectively), whereas si-3a2, si-3b1, si-3b3, and si-3b4 had no effect (Fig. 3A). Expression of si-1Y targeting eRF1 mRNA and corresponding to si1187, described by Carnes et al. (4), increased readthrough 2-fold, a level in good agreement with the results reported by the authors. Five days after electroporation, readthrough increased greatly (30-fold) for si-3a1 and si-3a9 (Fig. 3A) and only moderately for si-1Y and si-3a2 (8-fold and 4-fold, respectively). We did not detect any increase when using si-3b1, si-3b3, and si-3b4 targeting eRF3b mRNA. The kinetics of readthrough levels induced by siRNAs targeting eRF3a 3 to 7 days postelectroporation showed that the readthrough level reached a maximum after 5 to 6 days and then decreased (data not shown). In comparison, the readthrough level induced by siRNA directed against eRF3 reported by Janzen and Geballe (20) was only two- to threefold higher than the control level. This difference from our results could be explained by the fact that these authors measured readthrough only 2 to 3 days after transfection.
|
-tubulin antibodies as a loading control (a typical experiment is presented in Fig. 3B). A large decrease in eRF3a was observed for si-3a1- and si-3a9-expressing cells, in good correlation with the increase in readthrough levels. In comparison, si-3a2, which poorly affected readthrough, did not notably reduce eRF3a expression. As expected for specific siRNAs, si-3b1, si-3b3, and si-3b4, directed against eRF3b mRNA, did not change the expression level of eRF3a. The efficiency of eRF3b-targeted siRNAs and the absence of cross-response of eRF3a-targeted siRNAs on eRF3b expression were determined by Western blot analysis. Because endogenous eRF3b was not detectable in 293 cells, the cells were cotransfected with plasmid pCMV-heRF3b-GFP expressing an eRF3b-GFP fusion protein and plasmids expressing the siRNAs. The membrane was probed with anti-eRF3b antibodies, anti-neomycin antibodies as a control for the plasmid transfection level, and anti--tubulin as a loading control. The results show that siRNAs 3b1, 3b3, and 3b4 targeting eRF3b mRNA dramatically reduced the level of eRF3b-GFP protein, confirming their efficiency (Fig. 4). In contrast, siRNAs 3a1, 3a2, and 3a9 had no effect on eRF3b-GFP protein, demonstrating their specificity for eRF3a mRNA (Fig. 4). To verify the predicted interference of siRNAs with mRNA stability, the steady-state mRNA level was determined by Northern blot analysis for eRF3a and eRF1 mRNAs, with actin mRNA used as a loading control. As shown in Fig. 3D, the variations in eRF3a mRNA levels were similar in magnitude to those observed at the protein level: a strong reduction for si-3a1 and si-3a9, a moderate one for si-3a2, and no effect for siRNAs 3b1, 3b3, and 3b4.
|
In addition to its role in translation termination, eRF3 plays a role in other translation-coupled events, such as regulation of mRNA decay (17, 22) and recycling of ribosomes for translation initiation (8, 37). To determine whether these functions could influence the results of our readthrough assay, we examined the status of LacZ mRNA and of the encoded ß-galactosidase in siRNA-expressing cells. Northern blot and Western blot analyses indicated that LacZ mRNA (Fig. 3D) and truncated ß-galactosidase (Fig. 3B) levels remained unaffected by eRF3a depletion. Furthermore, the full-length ß-galactosidase resulting from stop codon readthrough was clearly visible in extracts of si-3a1- and si-3a9-electroporated cells (Fig. 3B).
Altogether, these results show that (i) eRF3a depletion promotes stop codon readthrough, demonstrating the involvement of eRF3a in translation termination; (ii) eRF3b silencing has no effect on readthrough, likely due to the absence or small amount of this protein in 293 cells; and (iii) eRF3a depletion induces a reduction of the eRF1 protein level, which probably contributes to increased readthrough. Finally, our results suggest that in 293 cells, eRF3a is the factor associated with eRF1 in the translation termination complex. However, we cannot rule out the possibility that eRF3b might be involved in translation termination in other cell types.
eRF3b alleviates the effect of eRF3a depletion on stop codon readthrough.
We next determined whether eRF3b could substitute for eRF3a in its role in translation termination and thus play the role of a release factor. For this purpose, eRF3a was depleted in 559C cells by electroporation of the plasmid expressing si-3a1, and 3 days later, the cells were reelectroporated with a set of pBK-CMV derivatives expressing either human eRF3a, human eRF3b, or their mutated forms carrying a double mutation in the G1 motif of the GTP-binding domain (see Materials and Methods). Three days after the second electroporation, the effect on stop codon readthrough was analyzed by measuring the ß-galactosidase activity in cell extracts. Cells electroporated with plasmid pSuper and reelectroporated with plasmid pBK-CMV served as a negative control (Fig. 5A, lane sup pBK). Cells electroporated with a plasmid expressing si-3a1 and reelectroporated with plasmid pBK-CMV were used as the standard level for readthrough promoted by eRF3a depletion (Fig. 5A, lane 3a1 pBK). For each electroporation experiment, we verified the amounts of endogenous eRF3a and eRF1 (Fig. 5B) and of the overexpressed release factors (Fig. 5B, eRF3a and eRF1, and C, eRF3b) by Western blot analysis. As expected, eRF3a overexpression induced a clear decrease in readthrough up to half of the standard readthrough level of 3a1/pBK (Fig. 5A, lane 3a1 3a). Most interestingly, eRF3b overexpression also induced a reduction of readthrough that was as great as that produced by eRF3a overexpression (lane 3a1 3b). However, we did not observe any reduction with the GTPase mutated forms of eRF3a and eRF3b (lanes 3a1 3aNN and 3a1 3bNN). These results showed that eRF3b can substitute for eRF3a in the translation termination process and point out the importance of their GTPase activities. Moreover, when looking at eRF1 levels by Western blotting (Fig. 5B), we observed that overexpression of eRF3a and eRF3b rescued the defect of eRF1 generated by eRF3a depletion. This rescue was only partial with both GTPase mutated forms, as shown by the eRF1/-tubulin ratio. Interestingly, this partial rescue did not induce a concomitant decrease in readthrough, suggesting a direct effect of eRF3a on termination efficiency.
|
Finally, we examined how eRF1 overexpression affected the readthrough level in eRF3a-depleted cells. As shown in Fig. 5A, overexpressed eRF1 induced only a moderate decrease in the readthrough level compared to the standard readthrough level, from 42-fold to 30-fold (lane 3a1 eRF1). This reduction in the readthrough level was likely due to the restoration of the eRF1 protein level to almost normal (Fig. 5B). At this basal level of eRF1, the remaining 30-fold increase in readthrough was likely directly promoted by eRF3a depletion alone. However, these experiments did not allow us to decide whether an excess of eRF1 can rescue a defect of eRF3a.
eRF3a depletion induces eRF1 protein degradation.
We have shown above that eRF3a depletion induced a reduction in the intracellular level of eRF1 protein (Fig. 3B) that was not due to a modification of the steady-state level of eRF1 mRNA (Fig. 3D). To further understand the mechanism involved in eRF1 reduction, we examined eRF1 protein stability in cells electroporated with si-3a1-expressing plasmid or pSuper as a control. For this purpose, 3 days after electroporation, cellular proteins were labeled by a 2-hour pulse with a mixture of [35S]methionine and [35S]cysteine and analyzed up to 72 h after being labeled by immunoprecipitation with anti-eRF1 and anti--tubulin antibodies (Fig. 6A). First, we observed that the amounts of eRF1 were almost the same for si-3a1-expressing cells and pSuper control cells after the 2-h labeling (Fig. 6B, time zero), suggesting that the translation of eRF1 was not affected by eRF3a depletion. Secondly, in si-3a1-expressing cells, there was a significant decrease of eRF1 protein as a function of time, which was not found in control cells (Fig. 6B). These results suggest that eRF1 is proteolytically degraded when not complexed with eRF3a, and they explain the reduction in eRF1 protein observed in eRF3a-depleted cells (Fig. 3B). However, with an eRF1 half-life of almost 40 h, this process seems to be very slow.
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have utilized specific antibodies directed against eRF3a or eRF3b to study their differential cellular expression. While eRF3a is expressed in all cell types tested with an abundance correlated with that of eRF1, eRF3b expression is restricted to brain tissue and varies during development. These results are in agreement with the tissue distribution of mouse eRF3a and eRF3b mRNAs reported by Hoshino et al. (15), showing by reverse transcription-PCR that eRF3b mRNA is present at lower levels than eRF3a mRNA in all tissues and is relatively enriched in the brain. Nevertheless, eRF3b cDNA was found in a wide variety of cDNA libraries, as shown by the numerous eRF3b sequences found in the GenBank EST collection. This could indicate that eRF3b protein may be expressed at a low level in all cell types, including 293 cells, but below the detection limits of our antibodies.
To determine the involvement of eRF3a and eRF3b in translation termination, we silenced eRF3a or eRF3b mRNA using siRNAs. Our results demonstrate unambiguously that eRF3a depletion induces a high level of stop codon readthrough whereas eRF3b silencing has no detectable effect. This suggests that in 293 cells, eRF3a plays a key role in translation termination efficiency and that eRF3b involvement, if any, was masked by the large amount of eRF3a. However, the demonstration that eRF3b can act as a release factor in vivo was obtained by overexpressing eRF3b in eRF3a-depleted cells (Fig. 5). Interestingly, the rescue of translation termination efficiency obtained with either eRF3a or eRF3b is roughly of the same magnitude. This suggests that eRF3b is as efficient as eRF3a in translation termination and that it must act in particular cell types or definite cellular states, which remain to be determined. As with eRF3a, we also observed a positive effect of eRF3b overexpression on the eRF1 level (Fig. 5B), confirming that eRF3b efficiently binds to eRF1 and restores its stability.
In addition, X. laevis eRF3, which is highly homologous to eRF3a and eRF3b in its C-terminal domain (96%) but closer to eRF3b than to eRF3a in its N-terminal extension, can also substitute for eRF3a. In contrast, neither the entire S. cerevisiae eRF3, which is highly divergent from human and Xenopus eRF3s in the N-terminal domain, nor the N-terminally truncated form of S. cerevisiae eRF3 carrying the conserved C-terminal domain only restores efficient termination (Fig. 5). However, in yeast, human eRF1 can complement yeast eRF1 mutants (38), showing that yeast eRF3 can associate with human eRF1 to form an active translation termination complex. Similarly, our results show that both forms of yeast eRF3 (entire and N-terminally truncated forms) rescue the eRF1 protein level (Fig. 5B), suggesting that they efficiently bind to human eRF1 but form an inactive complex. Interestingly, the overexpression of entire yeast eRF3 in eRF3a-depleted cells increases readthrough above the level obtained with si-3a1 alone, whereas the overexpression of the S. cerevisiae N-terminally truncated form does not. A possible explanation for this dominant-negative effect is that the long N-terminal extension of yeast eRF3 sequesters eRF1 in the inactive complex. These observations reinforce the previous suggestions (23, 25) that the N-terminal domain of eRF3 plays an important role in the formation of an active translation termination complex. The absence or improper conformation of the eRF3 N-terminal domain could possibly interfere either with the binding of the complex to the ribosome or with GTPase activity.
The same increase in readthrough above the level obtained with si-3a1 alone was found with eRF3a and eRF3b GTPase mutated forms, which carry a double mutation in the G1 motif of the GTPase domain (Fig. 5A). In addition, the results in Fig. 5B suggest that these mutated forms of eRF3 bind and stabilize eRF1, but less efficiently than wild-type eRF3a and eRF3b. The equivalent mutation in Ras GTPases (the S17N dominant-negative mutation) reduces the affinity for GTP, leading to the formation of a stable and inactive complex with the guanine exchange factor, but is insufficient to totally prevent the mutant form from binding GTP in vivo (9). As GTP binding seems to be required for eRF1-eRF3 association (22), we can speculate that our mutated forms of eRF3 bind GTP, though less efficiently than wild-type eRF3, and associate with eRF1 in an inactive complex.
In yeast, it has been reported recently that the reduction of the steady-state level of eRF3 does not modify eRF1 levels (33). In contrast, we show here that, in human cells, a depletion of eRF3a induces a significant reduction of the eRF1 level as observed by Western blotting (Fig. 3B). This reduction was further explained by a decrease in the eRF1 protein half-life (Fig. 6). In addition, overexpression of different forms of eRF3 alleviates eRF1 reduction (Fig. 5B). From these results, we hypothesize that eRF1 is proteolytically degraded when not complexed with eRF3. Thus, eRF1 degradation could allow the adjustment of its level to that of the eRF3 available for the formation of translation termination complexes, leading to the fine tuning of translation termination efficiency. This idea is reinforced when eRF3a and eRF1 expression levels in various human cell lines are compared, which shows that the abundance of eRF1 parallels that of eRF3a (Fig. 2A). However, the eRF1 protein seems to be very stable, and its mechanism of degradation is relatively slow. This is suggested by the pulse-chase experiment (Fig. 6) and the fact that, 5 days postelectroporation with si-3a1, only half of the eRF1 was degraded while eRF3a had almost completely disappeared (Fig. 3B). The high stability of eRF1 could explain the relatively inefficient silencing of eRF1 at the protein level in comparison with the almost complete knockdown of eRF3a. We also noticed that eRF1 depletion did not induce eRF3a reduction (Fig. 3C), suggesting that, due to its involvement in other cellular processes and association with factors such as PABP and Upf1, the intracellular amount of eRF3a is controlled by mechanisms other than termination complex formation.
In conclusion, in the present work, we have pointed out the importance of eRF3a in translation termination in mammalian cells: (i) eRF3a depletion induces an 30-fold increase in readthrough when a basal expression level of eRF1 is restored and (ii) eRF3a and eRF3b GTPase mutated forms and S. cerevisiae eRF3 overexpression in eRF3a-depleted cells restore eRF1 to a basal level without a concomitant decrease in readthrough. Both observations strongly suggest a direct effect of eRF3a on termination efficiency. Importantly, this work also gives prominence to the fact that eRF3a regulates the formation of the translation termination complex.
|
ACKNOWLEDGMENTS |
|---|
We thank Michel Philippe for critical reading of the manuscript.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Bourne, H. R., D. A. Sanders, and F. McCormick. 1991. The GTPase superfamily: conserved structure and molecular mechanism. Nature 349:117-127.[CrossRef][Medline]
3. Brummelkamp, T. R., R. Bernards, and R. Agami. 2002. A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550-553.
4. Carnes, J., M. Jacobson, L. Leinwand, and M. Yarus. 2003. Stop codon suppression via inhibition of eRF1 expression. RNA 9:648-653.
5. Chavatte, L., L. Frolova, L. Kisselev, and A. Favre. 2001. The polypeptide chain release factor eRF1 specifically contacts the s(4)UGA stop codon located in the A site of eukaryotic ribosomes. Eur. J. Biochem. 268:2896-2904.[Medline]
6. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159.[Medline]
7. Church, D. M., C. J. Stotler, J. L. Rutter, J. R. Murrell, J. A. Trofatter, and A. J. Buckler. 1994. Isolation of genes from complex sources of mammalian genomic DNA using exon amplification. Nat. Genet. 6:98-105.[CrossRef][Medline]
8. Cosson, B., N. Berkova, A. Couturier, S. Chabelskaya, M. Philippe, and G. Zhouravleva. 2002. Poly(A)-binding protein and eRF3 are associated in vivo in human and Xenopus cells. Biol. Cell 94:205-216.[CrossRef][Medline]
9. Feig, L. A. 1999. Tools of the trade: use of dominant-inhibitory mutants of Ras-family GTPases. Nat. Cell Biol. 1:E25-E27.[CrossRef][Medline]
10. Frolova, L., X. Le Goff, H. H. Rasmussen, S. Cheperegin, G. Drugeon, M. Kress, I. Arman, A. L. Haenni, J. E. Celis, M. Philippe, J. Justesen, and L. Kisselev. 1994. A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor. Nature 372:701-703.[CrossRef][Medline]
11. Frolova, L., X. Le Goff, G. Zhouravleva, E. Davydova, M. Philippe, and L. Kisselev. 1996. Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase. RNA 2:334-341.[Abstract]
12. Frolova, L. Y., J. L. Simonsen, T. I. Merkulova, D. Y. Litvinov, P. M. Martensen, V. O. Rechinsky, J. H. Camonis, L. L. Kisselev, and J. Justesen. 1998. Functional expression of eukaryotic polypeptide chain release factors 1 and 3 by means of baculovirus/insect cells and complex formation between the factors. Eur. J. Biochem. 256:36-44.[Medline]
13. Hegde, R., S. M. Srinivasula, P. Datta, M. Madesh, R. Wassell, Z. Zhang, N. Cheong, J. Nejmeh, T. Fernandes-Alnemri, S. Hoshino, and E. S. Alnemri. 2003. The polypeptide chain-releasing factor GSPT1/eRF3 is proteolytically processed into an IAP-binding protein. J. Biol. Chem. 278:38699-38706.
14. Hoshino, S., M. Imai, T. Kobayashi, N. Uchida, and T. Katada. 1999. The eukaryotic polypeptide chain releasing factor (eRF3/GSPT) carrying the translation termination signal to the 3'-poly(A) tail of mRNA. Direct association of eRF3/GSPT with polyadenylate-binding protein. J. Biol. Chem. 274:16677-16680.
15. Hoshino, S., M. Imai, M. Mizutani, Y. Kikuchi, F. Hanaoka, M. Ui, and T. Katada. 1998. Molecular cloning of a novel member of the eukaryotic polypeptide chain-releasing factors (eRF). Its identification as eRF3 interacting with eRF1. J. Biol. Chem. 273:22254-22259.
16. Hoshino, S., H. Miyazawa, T. Enomoto, F. Hanaoka, Y. Kikuchi, A. Kikuchi, and M. Ui. 1989. A human homologue of the yeast GST1 gene codes for a GTP-binding protein and is expressed in a proliferation-dependent manner in mammalian cells. EMBO J. 8:3807-3814.[Medline]
17. Hosoda, N., T. Kobayashi, N. Uchida, Y. Funakoshi, Y. Kikuchi, S. Hoshino, and T. Katada. 2003. Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation. J. Biol. Chem. 278:38287-38291.
18. Inge-Vechtomov, S. G., L. N. Mironova, and M. D. Ter-Avanesian. 1994. Ambiguity of translation: a eukaryotic version? Genetika 30:1022-1035.[Medline]
19. Jakobsen, C. G., T. M. Segaard, O. Jean-Jean, L. Frolova, and J. Justesen. 2001. Identification of a novel termination release factor eRF3b expressing the eRF3 activity in vitro and in vivo. Mol. Biol. 35:672-681.
20. Janzen, D. M., and A. P. Geballe. 2004. The effect of eukaryotic release factor depletion on translation termination in human cell lines. Nucleic Acids Res. 32:4491-4502.
21. Jean-Jean, O., X. Le Goff, and M. Philippe. 1996. Is there a human 
? C. R. Acad. Sci. Ser. III 319:487-492.[Medline]
22. Kobayashi, T., Y. Funakoshi, S. Hoshino, and T. Katada. 2004. The GTP-binding release factor eRF3 as a key mediator coupling translation termination to mRNA decay. J. Biol. Chem. 279:45693-45700.
23. Kong, C., K. Ito, M. A. Walsh, M. Wada, Y. Liu, S. Kumar, D. Barford, Y. Nakamura, and H. Song. 2004. Crystal structure and functional analysis of the eukaryotic class II release factor eRF3 from S. pombe. Mol. Cell 14:233-245.[CrossRef][Medline]
24. Larcher, J. C., L. Gasmi, W. Viranaicken, B. Edde, R. Bernard, I. Ginzburg, and P. Denoulet. 2004. Ilf3 and NF90 associate with the axonal targeting element of Tau mRNA. FASEB J. 18:1761-1763.
25. Le Goff, C., O. Zemlyanko, S. Moskalenko, N. Berkova, S. Inge-Vechtomov, M. Philippe, and G. Zhouravleva. 2002. Mouse GSPT2, but not GSPT1, can substitute for yeast eRF3 in vivo. Genes Cells 7:1043-1057.[Abstract]
26. Le Goff, X., M. Philippe, and O. Jean-Jean. 1997. Overexpression of human release factor 1 alone has an antisuppressor effect in human cells. Mol. Cell. Biol. 17:3164-3172.[Abstract]
27. Miccoli, L., A. Beurdeley-Thomas, G. De Pinieux, F. Sureau, S. Oudard, B. Dutrillaux, and M. F. Poupon. 1998. Light-induced photoactivation of hypericin affects the energy metabolism of human glioma cells by inhibiting hexokinase bound to mitochondria. Cancer Res. 58:5777-5786.
28. Otto, A. I., L. Riou, C. Marionnet, T. Mori, A. Sarasin, and T. Magnaldo. 1999. Differential behaviors toward ultraviolet A and B radiation of fibroblasts and keratinocytes from normal and DNA-repair-deficient patients. Cancer Res. 59:1212-1218.
29. Patino, M. M., J. J. Liu, J. R. Glover, and S. Lindquist. 1996. Support for the prion hypothesis for inheritance of a phenotypic trait in yeast. Science 273:622-626.[Abstract]
30. Paushkin, S. V., V. V. Kushnirov, V. N. Smirnov, and M. D. Ter-Avanesyan. 1997. Interaction between yeast Sup45p (eRF1) and Sup35p (eRF3) polypeptide chain release factors: implications for prion-dependent regulation. Mol. Cell. Biol. 17:2798-2805.[Abstract]
31. Paushkin, S. V., V. V. Kushnirov, V. N. Smirnov, and M. D. Ter-Avanesyan. 1996. Propagation of the yeast prion-like + determinant is mediated by oligomerization of the SUP35-encoded polypeptide chain release factor. EMBO J. 15:3127-3134.[Medline]
32. Phillips-Jones, M. K., L. S. Hill, J. Atkinson, and R. Martin. 1995. Context effects on misreading and suppression at UAG codons in human cells. Mol. Cell. Biol. 15:6593-6600.[Abstract]
33. Salas-Marco, J., and D. M. Bedwell. 2004. GTP hydrolysis by eRF3 facilitates stop codon decoding during eukaryotic translation termination. Mol. Cell. Biol. 24:7769-7778.
34. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
35. Stansfield, I., L. Eurwilaichitr, Akhmaloka, and M. F. Tuite. 1996. Depletion in the levels of the release factor eRF1 causes a reduction in the efficiency of translation termination in yeast. Mol. Microbiol. 20:1135-1143.[CrossRef][Medline]
36. Stansfield, I., K. M. Jones, V. V. Kushnirov, A. R. Dagkesamanskaya, A. I. Poznyakovski, S. V. Paushkin, C. R. Nierras, B. S. Cox, M. D. Ter-Avanesyan, and M. F. Tuite. 1995. The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. EMBO J. 14:4365-4373.[Medline]
37. Uchida, N., S. Hoshino, H. Imataka, N. Sonenberg, and T. Katada. 2002. A novel role of the mammalian GSPT/eRF3 associating with poly(A)-binding protein in Cap/Poly(A)-dependent translation. J. Biol. Chem. 277:50286-50292.
38. Urbero, B., L. Eurwilaichitr, I. Stansfield, J. P. Tassan, X. Le Goff, M. Kress, and M. F. Tuite. 1997. Expression of the release factor eRF1 (Sup45p) gene of higher eukaryotes in yeast and mammalian tissues. Biochimie 79:27-36.[Medline]
39. Weimer, T., J. Salfeld, and H. Will. 1987. Expression of the hepatitis B virus core gene in vitro and in vivo. J. Virol. 61:3109-3113.
40. Zhouravleva, G., L. Frolova, X. Le Goff, R. Le Guellec, S. Inge-Vechtomov, L. Kisselev, and M. Philippe. 1995. Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. EMBO J. 14:4065-4072.[Medline]
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|
