The v-erbA oncogene transforms chicken erythrocytic progenitors (T2EC) by blocking their differentiation and freezing them in a state of self-renewal. Transcriptomes of T2EC, expressing either v-erbA or a non-transforming form of v-erbA (S61G), were compared using serial analysis of gene expression and some, but not all, mRNA-encoding ribosomal proteins were seen to be affected by v-erbA. These results suggest that this oncogene could modulate the composition of ribosomes. In the present study, we demonstrate, using two-dimensional difference in gel electrophoresis, that v-erbA-expressing cells have a lower amount of RPL11 associated with the ribosomes. The presence of ribosomes devoid of RPL11 in v-erbA-expressing cells was further confirmed by immunoprecipitation. In order to assess the possible impact of these specialized ribosomes on the translational activity, we analyzed proteomes of either v-erbA or S61G-expressing cells using 2D/mass spectrometry, and identified nine proteins present in differing amounts within these cells. Among these proteins, we focused on HSP70 because of its involvement in erythroid differentiation. Our results indicate that, in v-erbA-expressing cells, hsp70 is not only transcribed but also translated more efficiently, as shown by polyribosome fractionation experiments. We demonstrate here, for the first time, the existence of ribosomes with different protein components, notably ribosomes devoid of RPL11, and a regulation of mRNA translation depending on v-erbA oncogene expression.
All hematopoietic cells are derived from the hematopoietic stem cells,1 which have a self-renewal capacity, that is, proliferating without losing their developmental potential or entering a differentiation pathway.2 The molecular mechanisms involved in the control of this self-renewal and of the differentiation process still remain elusive. Hence, the aim of our study focuses especially on the implication of the translational activity control in these processes. We have been studying these mechanisms using a cell model of primary avian erythrocytic progenitors named T2EC (TGF-α/TGF-β-induced erythrocytic cells) and the v-erbA oncogene.
T2EC were isolated from chicken bone marrow, which can either be maintained in a self-renewal state or induced to differentiate in vitro.3 Transformation of T2EC can be induced by infection with the avian erythroblastosis virus (AEV). This retrovirus, which carries the v-erbA and v-erbB viral oncogenes, induces erythroblastosis, carcinomas and sarcomas in chicken4 and transforms erythrocytic progenitors and chicken embryo fibroblasts in vitro.5 Expression of v-erbA alone is sufficient to stimulate the growth of chicken embryo fibroblasts,6 and to induce the transformation of erythrocytic progenitors by blocking their differentiation and freezing them in a state of self-renewal, both in vivo and in vitro.7, 8
v-erbA is derived from the c-erbAα1 proto-oncogene, which encodes the alpha form of the nuclear receptor for the thyroid hormone triiodothyronine.9 It is expressed as a gag/v-erbA fusion protein, which forms a heterodimeric complex with RXR. This complex recognizes the T3 response element and the retinoic acid response element, and represses the transcription of T3 and retinoic acid target genes, even in the presence of thyroid hormone T3 and retinoic acid.10 Hence, v-erbA was thought to act as a negative-dominant mutant of thyroid hormone triiodothyronine and retinoic acid receptor,11, 12 although we have recently demonstrated that the majority of v-erbA target genes were neither T3 nor retinoic acid-target genes.13 Moreover, the subcellular localization of v-erbA is different from that of c-erbAα1, which also confers on v-erbA other mechanisms of action.13, 14 Our previous works demonstrated that some of these mechanisms were related to the alteration of the mTOR and TGF-β signal transduction pathways in erythroid progenitors.15
In a previous study, transcriptomes of T2EC expressing either v-erbA or a non-transforming point mutant form of v-erbA, named S61G, were compared using serial analysis of gene expression (SAGE) and RT–qPCR (reverse transcription-quantitative polymerase chain reaction).13 Among the genes whose expression varies under the influence of v-erbA, several encode proteins implicated in translation, such as ribosomal proteins S3, S3a, S9, L13; translation initiation factors eIF4A, eIF5, eIF3 and EIF2B2-β; and the elongation factor eEF1-α1. Interestingly, only some, but not all, mRNA-encoding ribosomal proteins are expressed in decreased amounts in v-erbA-expressing cells. These results raised the intriguing possibility that v-erbA could modify the translational apparatus by modulating the ribosomal protein composition of ribosomes in chicken erythroid progenitors.
We report here, for the first time, the existence of ribosomes with different protein components in v-erbA-expressing cells and, specifically, of ribosomes devoid of RPL11. Moreover, we demonstrate the presence of translational regulation of HSP70 mRNA. All together, these results are compatible with the hypothesis according to which ‘specialized’ ribosomes could be involved in the translation of specific mRNA under the control of v-erbA oncogene.
v-erbA expression leads to ribosomes with a different composition of ribosomal proteins
SAGE analysis revealed reduced mRNA levels of some ribosomal proteins in v-erbA-expressing cells.13 To ensure that the total amount of ribosomes was not affected, the quantity of purified ribosomes from v-erbA- or S61G-expressing cells was determined by measuring the absorbance at 260 nm.16 Statistical analyses of data obtained from 18 independent experiments show no difference between the two cellular conditions (P-value: 0.6429) (Supplementary Data 1). This result clearly indicates that v-erbA expression in T2EC does not affect the overall number of ribosomes.
We therefore decided to analyze the ribosomal protein content in purified ribosomes using 2D-DIGE (two-dimensional difference in gel electrophoresis) in v-erbA- or S61G-expressing cells.
Ribosomal proteins associated with purified ribosomes were extracted, according to the procedure refined in our laboratory,16 and were labeled with CyDyes 3 and 5, respectively (Figure 1a). For the same biological sample, an inverted labeling was carried out (the flip-flop experiment). Four biological independent experiments were performed and the results were statistically analyzed with the t-test. The data indicated a difference in the amount of one ribosomal protein (P-value <0.05) between v-erbA- and S61G-expressing cells. Identification of this ribosomal protein was carried out, based on a previous characterization of Gallus gallus ribosomal proteins16 (Figure 1b), and it was identified as RPL11 (Figures 1c and d).
v-erbA expression in T2EC reduces RPL11 by 1.2-fold (P-value: 0.0412) (Figures 1c and d). This difference corresponds to about 100 000 ribosomes devoid of RPL11 in each cell of our model, and it is statistically significant and reproducible (obtained from eight different labeling experiments). These results strongly suggest the existence of ribosomes with different ribosomal protein components in v-erbA-expressing cells. More specifically, these data indicate that some ribosomes are devoid of RPL11 in T2EC infected with a retrovirus carrying the v-erbA oncogene.
Some ribosomes are devoid of RPL11 in v-erbA-expressing cells
In order to confirm the existence of ribosomes devoid of RPL11 in v-erbA-expressing cells by a different approach, ribosomes containing RPL11 were separated from ribosomes devoid of RPL11 by immunoprecipitation.
The purified ribosome pellets from cytosolic fractions cleared of mitochondria contain mostly entire ribosomes as demonstrated by the polyribosomal profile obtained from v-erbA- or S61G-expressing cells (Figure 5d).
These purified ribosome pellets were then incubated in the presence of an antibody directed against RPL11, then with G proteins bound to magnetic beads. Ribosomal particles containing RPL11 were separated from those devoid of RPL11 by magnetic sorting, the latter remaining in the supernatant (Figure 2a). Proteins contained in the fraction associated with the magnetic beads, and those present in the supernatant were analyzed by western blot. An antibody directed against RPL11 was used to measure the efficiency of the immunoprecipitation experiment (Figure 2b). In order to demonstrate the presence of ribosomes devoid of RPL11 in v-erbA-expressing cells compared to S61G-expressing cells, an antibody directed against RPL27, another ribosomal protein belonging to the same ribosomal subunit as RPL11, that is, the 60S ribosomal subunit, was applied (Figure 2c). Results obtained with anti-RPL27 clearly show a detectable number of ribosomes observed in the supernatant of v-erbA-expressing cells, but not in the supernatant from control cells (Figure 2c). This indicates that all the ribosomes from S61G-expressing cells do contain RPL11, but this is not the case for the ribosomes from v-erbA-expressing cells, which show a detectable amount of RPL11-devoid ribosomes. The ratios of the signals obtained for RPL11 and RPL27 in the supernatants were then calculated and compared for v-erbA- and S61G-expressing cells. Results from two independent experiments clearly show a mean RPL11/RPL27 ratio in the supernatants that is lower for v-erbA-expressing T2EC than for S61G-expressing T2EC (ratio 1: 0.194 (experiment shown in Figure 2); ratio 2: 0.21 (data not shown).
Consequently, by two different approaches (2D-DIGE and immunuprecipitation), our results demonstrate the presence of ribosomes exhibiting a difference in the composition of ribosomal proteins between v-erbA- and S61G-expressing cells: in v-erbA-expressing cells, a detectable proportion of ribosomes appear to be devoid of RPL11. These data provide the first evidence that the v-erbA oncogene does modulate the protein content of ribosomes, and raises the intriguing possibility that this might affect the translational apparatus.
Presence of RPL11 non-associated to ribosomes in v-erbA-expressing cells
We then wanted to assess whether the presence of ribosomes devoid of RPL11 resulted from a global downregulation of RPL11 amount. For this, total proteins were analyzed by western blot (Figure 3a). The results from seven independent experiments were then compared using a t-test (Figure 3b). Unexpectedly, they showed that RPL11 was slightly more abundant in v-erbA than in S61G-expressing cells (P-value: 0.02). A similar increase has been detected at the mRNA level by RT–qPCR (1.2 fold; data not shown).
These results suggest that ribosomes devoid of RPL11 do not arise through a deficiency of rpl11 expression but rather from a ribosome-dependent mechanism. Furthermore, this indirectly suggests that RPL11 non-associated to ribosomes is slightly more abundant in v-erbA- than in S61G-expressing cells. This RPL11 non-associated to ribosomes could exhibit extra-ribosomal functions potentially involved in the cellular transformation process. Mechanisms implicating extra-ribosomal functions of RPL11 will be not explored further in this study. We will focus here more specifically on the possible implication of RPL11 in the translational process.
Identification of proteins, the expression of which differs under v-erbA expression
In order to explore the potential involvement of v-erbA oncogene in the translation process, we performed a comparison of cytoplasmic and nuclear proteomes of either v-erbA- or S61G-expressing cells using 2-DE (two-dimensional gel electrophoresis).
Cytosolic and nuclear proteins were extracted, separated by 2-DE and then the proteins were silver stained. The levels of proteins in both conditions were then compared using the Progenesis SameSpots software (Nonlinear Dynamics, Newcastle upon Tyne, UK) (Figures 4a and b). Data from four independent experiments were analyzed using the t-test. The results show that six cytosolic and three nuclear proteins exhibit different amounts (P-value <0.1), three of which reach the statistical significance level (P-value <0.05). Proteins were identified by mass spectrometry (Figures 4c and d). Three of the cytosolic proteins were identified as 78 kDa glucose regulated protein, heat shock protein 70 and L-lactate dehydrogenase B chain; the three other cytosolic proteins identified still have no attributed functions, but they are homologs of FKBP4, SYNCRIP and ACAT1; those identified in the nuclear fraction were GTP-binding nuclear protein RAN, β-actin and Syndesmos.
Contrary to the data obtained with SAGE and by RT–qPCR that showed only a repression of gene expression by v-erbA, data resulting from the proteomic comparison show both a decrease (for homologs of FKBP4, homolog of SYNCRIP, homolog of ACAT1, LDHB, Ran chick and β-actin) and an increase (for GRP78, HSP70 and Syndesmos) in the amounts of some proteins in v-erbA-expressing cells. These results suggest that gene expression of the identified proteins may be subject to transcriptional and/or translational regulations by the v-erbA oncogene.
In order to clarify this point, the total mRNA encoding these proteins, from either v-erbA- or S61G-expressing cells, was analyzed by RT–qPCR. The total amounts of mRNA from five proteins showed no difference (homolog of ACAT1, Ran chick, Syndesmos, β-actin and GRP78) while three others were present in lower amounts (homolog of FKBP4 (0.90), homolog of SYNCRIP (0.94) and LDHB (0.88) in v-erbA-expressing cells (data not shown). These results cannot be directly compared with those obtained by 2-DE because, on the one hand, we analyzed total mRNA levels and, on the other hand, we acquired proteomic data obtained from cytoplasmic or nuclear fractions because the analysis of total cellular protein content was too complex. However, these data point towards the involvement of the v-erbA oncogene in the regulation of the translational process.
In order to get a much clearer picture of possible regulations affecting given v-erbA targets, we decided to focus on the hsp70 gene expression; although it did not exhibit the best P-value, its biological function made it a promising candidate: HSP70 is a chaperone protein, which favors the erythroid differentiation process by protecting GATA-1 from degradation by caspases.17
v-erbA oncogene regulates expression of the hsp70 gene at both the transcriptional/posttranscriptional and translational levels
Results obtained from RT–qPCR assays showed that v-erbA expression can induce a 1.36-fold (P-value: 0.019) increase in the amount of HSP70 mRNA in T2EC, which could be due to the transcriptional/posttranscriptional regulations (Figure 5a).
To test whether the v-erbA oncogene could also regulate the translation of HSP70 mRNA, we first analyzed the total cell content in HSP70 by western blot (Figure 5b). Results from seven independent experiments were compared using the Wilcoxon test, and they indicated a 2.43-fold increase in the amount of HSP70 protein (P-value: 0.078) in v-erbA-expressing cells (Figure 5c). The variability observed here is well known in our cell model and it was highlighted in other publications from our laboratory.13 We suspect it might very well be related to the known stochasticity in gene expression,18 reinforced by T2ECs being primary cells, stemming from different bone marrow extractions.
Overall, the results obtained with RT–qPCR correlate with those obtained by western blot. However, the level of fold induction of the HSP70 protein is twice higher than that of HSP70 mRNA (2.43 versus 1.36) (Figures 5c and a), suggesting the existence of a possible additional regulation of this gene by the v-erbA oncogene. This increase in HSP70 amount could be due to a post-translational regulation (like a longer half-life of HSP70) and/or due to a translational regulation (like an increase in synthesis of this protein).
Consequently, we analyzed the HSP70 mRNA distribution in different polyribosomal fractions. We separated fractions containing non-translated mRNA, mRNA committed to the translational process, and strongly translated mRNA (Figure 5d). Data obtained in the three polyribosomal fractions with the three reverse transcription assays of two independent experiments were statistically analyzed with the t-test; the P-values calculated were 0.027, 0.063 and 0.0001 for the fractions containing non-translated mRNA, mRNA committed to the translational process, and highly translated mRNA, respectively.
Two features were readily apparent in v-erbA-expressing cells: (1) a general increase in the amount of HSP70 mRNA, supporting the results of experiments carried out on total mRNA (Figure 5a), and (2) a clear over-representation of HSP70 mRNA in fractions containing highly translated mRNA. Data obtained by polysomal comparison show a significant translational regulation of HSP70 mRNA under the influence of v-erbA oncogene, as evidenced by a much higher engagement rate of the mRNA in the translational apparatus.
Data resulting from RT–qPCR assays, western blot and RT–qPCR associated with polyribosomal analysis clearly demonstrate a regulation in hsp70 gene expression both at the transcriptional/posttranscriptional and at the translational level and, thus, confirm the change in the activity of the translational apparatus, in T2EC, induced by the v-erbA oncogene.
V-erbA oncogene blocks the differentiation process of chicken erythroid progenitors, preventing the exit from their self-renewing state. In an attempt to elucidate its mechanism of action, a previous transcriptomic analysis demonstrated that, among other v-erbA target genes, one could find some, but not all, of the mRNA-encoding ribosomal proteins. We therefore carried out a more detailed analysis of the effect of v-erbA on ribosomal proteins and ribosomes. The main conclusions of the present study are as follows:
2D-DIGE and immunoprecipitation experiments demonstrated, by two independent approaches, the existence of ribosomes devoid of RPL11 in v-erbA-expressing cells.
Among the target proteins of v-erbA identified by 2DE, we focused on HSP70 and demonstrated a significant increase in the recruitment of its mRNA by the translational apparatus in v-erbA-expressing cells.
Since studies carried out in yeast showed that RPL11 is essential for the 60S subunit maturation, as well as for their cytoplasmic exportation,19, 20 one wonders how ribosomes devoid of RPL11 could exist in the cytoplasm. It is possible that RPL11 would be associated with the 60S subunit during the first steps of ribosomal maturation. Then, in the cytoplasm and after the binding of other ribosomal protein(s) onto the rRNA, RPL11 could become detached, since it has been observed during ribosome biogenesis that the binding of a new ribosomal protein on rRNA can induce conformational changes of the latter.21 Alternatively, it is possible that post-transcriptional modifications to the rRNA could induce RPL11 loss in the cytoplasm. These hypotheses predict the existence of a pool of RPL11 non-associated to ribosomes in v-erbA-expressing cells, which is indeed supported by our findings, since we have shown that the global level of RPL11 increases slightly under v-erbA expression. We can imagine that this pool of RPL11 non-associated to ribosomes, in the erythrocytic progenitors, could become associated with different complexes in order to promote its extra-ribosomal functions. Diverse extra-ribosomal functions of RPL11 have been identified in recent years, such as its involvement in the MDM2–p53 pathway22, 23 and in the transcriptional24 and translational25 expression of the c-myc oncogene.
Overall, our data suggest the following possibility: in v-erbA-expressing erythrocytic progenitors, different populations of ribosomes, particularly ribosomes devoid of RPL11, do coexist and could favor the translation of a specific set of mRNA, such as HSP70 mRNA, although the direct link between the presence of specialized ribosomes and specific mRNA translation under the control of the v-erbA oncogene still needs to be formally demonstrated. Altered translation of these specific mRNAs could participate in maintaining T2EC in a self-renewal state by blocking their commitment towards differentiation, together with the many previously identified mechanisms of action spanning transcriptional regulation13, 26 and signaling regulation.15 Moreover, our previous studies showed that the mTOR signaling pathway was activated under the influence of v-erbA,15 which could notably favor an important translation of 5′TOP mRNA.27 Consequences observed on the translational regulation of some mRNA under the control of v-erbA, particularly HSP70 mRNA, in the work related here, could thus result both from the presence of ribosomes devoid of RPL11 and/or the activation of the mTOR signaling pathway.
Among the putative genes that may be subjected to translational regulation by v-erbA, we focused on the hsp70 gene and demonstrated the involvement of the v-erbA oncogene in regulating the translation of its mRNA. HSP70 is a very interesting protein target, because it is a chaperone protein that participates in different biological processes, including apoptosis inhibition. HSP70 has been shown to behave as an anti-apoptotic protein, which is strongly and preferentially expressed in human tumor cells.28, 29 Expression of hsp70 has been correlated with cellular proliferation, low differentiation, metastasis development and negative therapeutic results in breast cancers.30, 31 In erythrocytic progenitors, in the presence of erythropoietin, HSP70 adopts a nuclear localization and protects the transcription factor GATA-1 (erythroid transcription factor) from caspase-3 cleavage.32 GATA-1 is essential for activating erythroid gene expression, as well as the expression of apoptosis-inhibiting genes such as bclxl.33 In v-erbA-expressing erythrocytic progenitors, HSP70 might, therefore, participate in the apoptosis-inhibiting mechanisms, although its putative role in a differentiation blockade seems more elusive.
The possibility that ‘specialized ribosomes’, whose composition might not be the canonical one, could exist is becoming increasingly recognized as an important mechanism in the regulation of gene expression (see Xue and Barna34 for a recent review). In E. coli, in the presence of the Kasugamycin antibiotic, specific mRNA can be translated by 61S ribosomal particles. These ‘specialized’ and functional ribosomes are devoid of six ribosomal proteins: S1, S1, S6, S12, S18 and S21.35 Komili et al.36 and Cardenas et al.37 have also shown the correlation of heterogeneous ribosome presence, containing different paralogs, in yeast with the translation of specific mRNA and the regulation of protein synthesis efficiency and growth rate.36, 37 Furthermore, different ribosomal paralogs, displaying non-redundant functions, exhibit a differential level of expression in Drosophila melanogaster testes, suggesting the need for a specific translational apparatus and, in particular, specialized ribosomes for germ cell development.38 Other data indicated the presence of specific-tissue ribosomal proteins: RPL10-like and RPL39-like proteins were found only in testes, whereas the RPL22-like protein was absent from testes but present in liver and mammary tissue.39 Altogether these data demonstrate the presence of ribosomes with different ribosomal protein contents associated with specific phenotypes in various species. However, none of them demonstrated until now the direct physical link between the specialized ribosomes and these particular sets of mRNA. Consequently, this regulation process needs to be investigated in closer detail in physiologically relevant contexts.
Studies performed on various species indicated yet the involvement of specialized ribosomes in the control of physiological processes. However, our results underline the importance of the translational activity control in pathological processes as well. In our avian model, we have demonstrated the existence of specialized ribosomes in v-erbA-transformed cells and this, in turn, indicates the possible presence of miscellaneous ribosomal populations, with different ribosomal protein contents, in human cancers. This subject warrants more detailed studies, especially regarding the possible involvement of these specialized ribosomes in cellular transformation processes, possibly via the translation of specific set of mRNA.
Materials and methods
Cell cultures and viral infections
T2EC were expanded as already described.3 T2EC were infected with a viral supernatant from XJ126, 40 and S61G41, 42 and maintained, for 5 days, in the presence of 3 mg/ml G418 (Gibco/Life Technologies SAS, St Aubin, France).
Cell lysis and fractionation: nuclear proteins, cytosolic proteins and ribosomal proteins
T2EC were harvested in lysis buffer as described.16 The cell lysate was centrifuged at 750 g for 10 min, at 4 °C, to spin down the nuclei. Nuclei were re-suspended in the buffer containing 50 mM Tris-HCl pH 7.4, 25 mM KCl, 5 mM MgCl2, and 2 mM DTE. Nuclear proteins were extracted as described43, 44 and then dissolved in acetic acid. They were lyophilized and then purified with the 2-D cleanup kit (GE Healthcare, Pittsburgh, PA, USA) and, finally, solubilized in 8 M urea, 2% CHAPS, 0.001% bromophenol blue, 0.5% IPG buffer pH 3–10 (GE Healthcare, Europe GmbH, Velizy-Villacoublay, France)), and 40 mM DTE.
Post-nuclear supernatant was centrifuged at 12 000 g for 10 min, at 4 °C, to spin down the mitochondria. The post-mitochondrial supernatant was treated with two different protocols in order to obtain the cytosolic protein fraction or purified ribosomes, respectively.
The cytosolic protein fraction was acquired after the purification of the post-mitochondrial supernatant with the 2D cleanup kit and proteins were then re-suspended in the same rehydratation buffer as described for the nuclear protein fraction.
To obtain purified ribosomes, post-mitochondrial supernatant was layered on 1 ml of a 1 M sucrose cushion in a centrifuged tube, and centrifuged for 2 h at 305 000 g at 4 °C. Purified ribosome pellet was resuspended in a buffer containing 50 mM Tris–HCl pH 7.4, 25 mM KCl, 5 mM MgCl2 and 2 mM DTE.16 Concentration of the total amount of purified ribosomes was determined by measuring the absorbance at 260 nm with a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). At this wavelength, 14 OD corresponds approximately to 1 mg of ribosomes.45
Ribosomal protein extraction, reduction and alkylation steps were performed according to the previously published protocol.16
2-DE, the silver staining method and protein identification
For v-erbA- or S61G-expressing cells, the same amount of proteins was used for the 2-DE analysis. Between 200 and 450 μg of cytosolic proteins, or 350–650 μg of nuclear proteins, were re-suspended in rehydration buffer and analyzed by 2-DE as already described.23
Proteins were silver stained according to the published protocol.46 Differentially expressed proteins were identified using the statistical tools in Progenesis Stats (Nonlinear Dynamics, Durham, NC, USA). Analysis of variance was applied to matched spots, and the data were filtered to retain protein spots with P⩽0.1, determined by 1-way analysis of variance. Proteins of interest were cut out from dried gels and identified by mass spectrometry as described in the published protocol47 with minor modifications: consecutive searches against the Swiss-Prot-TrEMBL_decoy (Chordata taxonomy) and contaminants databases (3311496 sequences in total) were performed for each sample using Mascot (version 2.3).
Two-Dimensional Difference in Gel Electrophoresis
Ribosomal proteins were labelled with CyDye DIGE Fluors following the manufacturer’s instructions (GE Healthcare). For the same biological sample, the inverted labelling was systematically performed.
Labelled ribosomal proteins were analyzed by 2-DE as already described.16 Gels were scanned using the Typhoon Trio variable-mode imager (GE Healthcare). Image analysis was performed using the Progenesis SameSpots Software. Differentially expressed ribosomal proteins were identified and analyzed as described for nuclear and cytosolic proteins.
Co-immunoprecipitation and immunoblotting experiments
For immunoprecipitation, polyribosomes from T2EC, expressing either S61G or v-erbA, were extracted as described in the paragraph ‘ribosomal protein fraction’, using buffers without DTE. Ten microgram of ribosomes were incubated overnight, at 4 °C, in the presence of 2 μg specific anti-RPL11 primary antibody (FL-178, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The next day, 100 μl of PureProteome Protein G coupled to magnetic beads (Millipore, Billerica, MA, USA) were added to ribosomes for 2 h at 4 °C. These ribosomes were then submitted to a magnetic sorting, following the manufacturer’s instructions (Millipore), allowing for the separation of ribosomes with RPL11 associated with the magnetic beads from the ribosomes devoid of RPL11 contained in the supernatant. Ribosomal proteins contained in the supernatant or associated to the beads were analyzed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and probed with antibodies directed against RPL11 and RPL27 (Sigma-Aldrich, St Quentin-Fallavier, France).
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blot analysis
Different quantities of proteins per sample were subjected to western blot analysis using anti-RPL11 antibody (1:1000), anti-RPL27 antibody (1:2500), anti-HSPA2 antibody (1:10000; Epitomics, Burlingame, CA, USA, #3262-S) and GAPDH antibody (1:35000; Cell Signaling Technology, Danvers, MA, USA, #2118). Secondary HRP-conjugated mouse (Cell Signaling Technology, #7076) or rabbit (Cell Signaling Technology, #7074) antibodies were used at a dilution of 1:10 000. Membranes were developed using the enhanced chemiluminescence system (GE Healthcare). Protein quantification was performed with the Image J software (Research Services Branch (RBS), Image Processing and Analysis in Java (http://rsb.info.nih.gov)).
Data passing the Shapiro–Wilk normality test were analyzed either by Student test for the normalized values or Wilcoxon test for the non-normalized values with the R statistical software. Data were filtered to retain results with P-value ⩽0.05.
Distribution of specific mRNA among polyribosomal fractions
Polyribosomes were prepared from 80 × 106 cells. Fractionation of polyribosomes on 10–40% sucrose gradient was carried out from post-mitochondrial supernatants as previously described.48 Fifteen fractions of 750 μl were collected from the top of the gradient with continuous monitoring of absorbance at 254 nm. The first two fractions were eliminated because they contained only small contaminant particles. Each of the 13 remaining fractions was treated with 100 μg/ml proteinase K, 2% sodium dodecyl sulfate, and 1 unit/μl RNAsine, prior to phenyl–chloroform extraction and ethanol precipitation of the RNA.
Extraction of total mRNA and real-time RT–PCR
Total RNA was extracted with the RNeasy Mini Kit (QIAGEN, Hilden, Germany), followed by a DNAse treatment (Ambion, Austin, TX, USA). Reverse transcription (RT) assays were performed on 500 ng of total RNA with the Super Script III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Real-time PCR experiments used the MX 3000 PRO (Stratagene, Kirkland, WA, USA) with the QuantiTect SYBR Green PCR Kit (QIAGEN) or the Coffrets KAPA SYBR FAST qPCR (CliniSciences, Nanterre, France).
Specific primers were used to quantify the expression of Heat Shock Protein 70 (Hsp70; UniProtKB accession number: B3VHV2).
Standard invariant genes used for quantification of HSP70 total mRNA were ATP synthase subunit B (UniProtKB accession number: Q5ZLC5), T complex protein 1 (beta unit) (NCBI accession number: NP_001012551.1), GAPDH (GenBank accession number: K01458) and hnRNP (Sigenae accession number: AB038184.108.40.206). ATP synthase subunit B was used as a standard invariant gene in the polyribosomal experiments.
For each pair of primers, the efficiency of PCR was determined using a standard curve generated by a dilution series of the sample that had the highest expression rate for the selected gene. The global quantification for each gene was based on the published mathematical formula.49
For information regarding the specific PCR conditions and primer sequences used, please contact the authors.
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We thank Dr Stephanie Gobert-Gosse (Universite Claude Bernard Lyon 1, Villeurbanne, France) for her expert advice on the two-dimensional electrophoresis experiments and Clément Soleilhavoup for his technical help in the analysis of total mRNA levels by RT–qPCR. This work received support from grants from Association pour la Recherche contre le Cancer, Ligue contre le Cancer (Comité Départemental du Rhône), Société Française d’Hématologie (SFH), Région Rhône-Alpes, Université Claude Bernard Lyon 1 (UCBL1) and Centre National de la Recherche Scientifique (CNRS).
Supplementary Information accompanies this paper on the Oncogene website
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Nguyen-Lefebvre, A., Leprun, G., Morin, V. et al. V-erbA generates ribosomes devoid of RPL11 and regulates translational activity in avian erythroid progenitors. Oncogene 33, 1581–1589 (2014). https://doi.org/10.1038/onc.2013.93
- v-erbA oncogene
- ribosomal proteins
- translational activity regulation
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