CPEB2-eEF2 interaction impedes HIF-1α RNA translation
2011; Springer Nature; Volume: 31; Issue: 4 Linguagem: Inglês
10.1038/emboj.2011.448
ISSN1460-2075
Autores Tópico(s)Cancer-related molecular mechanisms research
ResumoArticle9 December 2011free access Source Data CPEB2–eEF2 interaction impedes HIF-1α RNA translation Po-Jen Chen Po-Jen Chen Division of Neuroscience, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan National Defense Medical Center, Graduate Institute of Life Sciences, Taipei, Taiwan Search for more papers by this author Yi-Shuian Huang Corresponding Author Yi-Shuian Huang Division of Neuroscience, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan National Defense Medical Center, Graduate Institute of Life Sciences, Taipei, Taiwan Search for more papers by this author Po-Jen Chen Po-Jen Chen Division of Neuroscience, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan National Defense Medical Center, Graduate Institute of Life Sciences, Taipei, Taiwan Search for more papers by this author Yi-Shuian Huang Corresponding Author Yi-Shuian Huang Division of Neuroscience, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan National Defense Medical Center, Graduate Institute of Life Sciences, Taipei, Taiwan Search for more papers by this author Author Information Po-Jen Chen1,2 and Yi-Shuian Huang 1,2 1Division of Neuroscience, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 2National Defense Medical Center, Graduate Institute of Life Sciences, Taipei, Taiwan *Corresponding author. Institute of Biomedical Sciences, Academia Sinica, 128 Section 2, Academia Road, N703 Taipei 11529, Taiwan. Tel.: +886 22652 3523; Fax: +886 22785 8594; E-mail: [email protected] The EMBO Journal (2012)31:959-971https://doi.org/10.1038/emboj.2011.448 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Translation of mRNA into protein proceeds in three phases: initiation, elongation, and termination. Regulated translation allows the prompt production of selective proteins in response to physiological needs and is often controlled by sequence-specific RNA-binding proteins that function at initiation. Whether the elongation phase of translation can be modulated individually by trans-acting factors to synthesize polypeptides at variable rates remains to be determined. Here, we demonstrate that the RNA-binding protein, cytoplasmic polyadenylation element binding protein (CPEB)2, interacts with the elongation factor, eEF2, to reduce eEF2/ribosome-triggered GTP hydrolysis in vitro and slow down peptide elongation of CPEB2-bound RNA in vivo. The interaction of CPEB2 with eEF2 downregulates HIF-1α RNA translation under normoxic conditions; however, when cells encounter oxidative stress, CPEB2 dissociates from HIF-1α RNA, leading to rapid synthesis of HIF-1α for hypoxic adaptation. This study delineates the molecular mechanism of CPEB2-repressed translation and presents a unique model for controlling transcript-selective translation at elongation. Introduction Translation of mRNA into protein is coordinated by the interplay of ribosomes and general translation factors. This process is composed of the following three phases: (1) Initiation: The sequential assembly of eukaryotic initiation factors (eIFs) and the 40S ribosome at the 5′ end of RNA forms the 48S complex that scans the 5′-untranslated region (5′-UTR) until it locates the AUG start codon. Upon the release of eIFs and joining of the 60S ribosomal subunit, the translation-competent 80S ribosome is assembled at the start codon ready for the next step. (2) Elongation: The open reading frame of the mRNA is decoded by the repetitive and coordinated actions of the 80S ribosome, eukaryotic elongation factors (eEFs) and aminoacyl-charged tRNAs to synthesize a specific polypeptide chain until the entire coding sequence is translated and a stop codon is reached. (3) Termination: Translation ceases in this final stage. Once the eukaryotic releasing factors (eRFs) bind to the stop codon, they prompt release of the polypeptide chain and disassembly of the entire ribosome–mRNA complex to conclude one round of translation (Merrick and Nyborg, 2000; Mathews et al, 2007; Pestova et al, 2007). In addition to the general synthesis machinery, translation of selective mRNAs can be modulated with cis-regulatory sequences that often reside in the 5′- or 3′-UTR of the mRNA and/or with their cognate RNA-binding proteins. Virtually, all RNA-binding proteins identified to date mechanistically regulate translation at the initiation stage (Richter and Sonenberg, 2005; Sonenberg and Hinnebusch, 2009). In contrast, polypeptide elongation is usually modulated in a general manner through phosphorylation of eEF2 to impair its binding to GTP, thereby decreasing the rate of peptide elongation and contributing to global inhibition of protein synthesis (Carlberg et al, 1990; Nygard et al, 1991). Although polysome profiling experiments indicate that the translational regulation of several transcripts likely occurs at elongation (Olsen and Ambros, 1999; Clark et al, 2000; Waerner et al, 2006; Galban et al, 2008), the mechanisms underlying these observations have just begun to surface with a recent report showing that the heterogeneous nuclear ribonucleoprotein (hnRNP) E1 inhibits the release of eEF1A1 from ribosomes and stops translation elongation of its target RNAs (Hussey et al, 2011). The CPEB (cytoplasmic polyadenylation element binding protein)-like proteins, CPEB2, CPEB3, and CPEB4, were identified because they showed a similar structure and sequence in the C-terminal RNA-binding regions to CPEB1 (Mendez and Richter, 2001). CPEB1 represses translation via binding to the eIF4E-binding protein, maskin, or neuroguidin, which blocks translation initiation by interfering with the assembly of eIF4E and eIF4G (Stebbins-Boaz et al, 1999; Jung et al, 2006). Although the previous studies show that CPEB2 and CPEB3 repress target RNA translation (Huang et al, 2006; Hagele et al, 2009), the molecular mechanisms accounted for this inhibition have not been revealed. In the present study, using a yeast two-hybrid screen and co-immunoprecipitation (co-IP) assays, we found that CPEB2 directly interacted with the elongation factor, eEF2, for which guanosine triphosphatase (GTPase) activity is induced upon binding to ribosomes and required for peptide translocation (Merrick and Nyborg, 2000; Hartman and Smith, 2010). Using an in-vitro reconstituted system (Iwasaki and Kaziro, 1979), the rate of eEF2/ribosome-activated GTP hydrolysis was diminished by CPEB2. In the tethered function assay, CPEB2 inhibited the reporter RNA translation only when binding to the RNA. Such repression persisted in eIF-independent translation (Wilson et al, 2000; Pestova and Hellen, 2003) and was sensitive to an agent that blocks elongation, but not initiation. Moreover, CPEB2 in which the eEF2-interacting motif had been deleted lost its repressor function; thus, CPEB2 impedes target RNA translation at elongation. The only known target of CPEB2 is hypoxia-inducible factor-1α (HIF-1α) RNA, which encodes a transcription factor that regulates several hypoxia-inducible genes. HIF-1α is constantly synthesized, prolyl-hydroxylated and degraded in the well-oxygenated environment; however, in response to hypoxia- or chemical-induced oxidative stress, the HIF-1α level is rapidly elevated due to an increase in translation and blockade of degradation (Yee Koh et al, 2008; Majmundar et al, 2010). Several polysomal profiling studies have reported that elevated HIF-1α synthesis is concomitant with the migration of HIF-1α RNA from polysomes of light density towards polysomes of heavy density (Hui et al, 2006; Thomas and Johannes, 2007; Galban et al, 2008), suggesting that upregulated HIF-1α synthesis during hypoxia may be first contributed by increasing the translation efficiency of HIF-1α RNA that are already in the elongation phase. Despite much attention is paid to investigate HIF-1α synthesis under hypoxia, it has not been assessed whether HIF-1α RNA is subject to translational control under normoxia since HIF-1α protein is degraded and barely detectable in most cells. Here, we found that the interaction between CPEB2 and eEF2 slowed down translation of HIF-1α RNA; however, arsenite-induced oxidative stress caused the dissociation of CPEB2 from HIF-1α RNA, resulting in augmentation of HIF-1α synthesis. Taken together, our study reveals the molecular mechanism underlying CPEB2-repressed translation. Notably, the CPEB2–eEF2 interaction represents a unique example in which the peptide elongation rate from individual RNA is modulated through a 3′-UTR-bound translational repressor to control the rate-limiting step of protein synthesis at elongation. Results Identification and expression analysis of novel CPEB2 isoforms A previous study using northern blotting showed that CPEB2 mRNA was expressed at high levels in the testes and brain (Theis et al, 2003); however, the tissue distribution of CPEB2 protein has not been examined. Because CPEB2 shares 95% sequence identity with CPEB3 and CPEB4 in the C-terminal RNA-binding domain, we used the N-terminal 261 amino acids (a.a.) of mouse CPEB2 (NP_787951, 521 a.a.) as the immunogen to generate a CPEB2-specific antibody that did not recognize other CPEB proteins (Supplementary Figure S1). This affinity-purified antibody showed that CPEB2 proteins from neurons migrated at about 100 and 135 kDa on SDS–polyacrylamide gel (PAGE), which were larger than the published mouse sequence (Figure 1A). Because the immunostained signals were diminished in CPEB2 knockdown (KD) neurons (Figure 1A), the NP_787951 clone is unlikely to contain full-length CPEB2. To identify the longer transcripts, primers designed according to the predicted rat CPEB2 sequence (XM_001060239, 724 a.a.) were used to amplify the coding region from hippocampal neuron cDNA. Two unreported alternatively spliced sequences, CPEB2a and CPEB2b, were isolated and deposited in the NCBI database, JF973322 and JF973323, respectively (Figure 1B). CPEB2a and CPEB2b, when co-expressed in Neuro-2a cells, migrated at a similar position to endogenous CPEB2 of 100 kDa on SDS–PAGE (Figure 1C). Notably, a weak signal of ∼135 kDa was also detected (Figure 1C). This 135 kDa isoform (NP_787951.2) was recently deposited to replace the original NP_787951; however, most CPEB2 from neurons and Neuro-2a cells appears to be encoded by CPEB2a and CPEB2b sequences. A comparison of the genomic organization of CPEB2a, CPEB2b, and NP_787951.2 is illustrated in Figure 1D. Because the antibody was raised against the common region of all isoforms, the tissue distribution of CPEB2 was examined by western blotting. Except in the testes where the 135-kDa isoform was abundantly expressed, the predominant forms in other tissues appear to be CPEB2a/2b (Figure 1E). Figure 1.Identification and expression analysis of CPEB2 isoforms. (A) CPEB2 proteins in the control (siCtrl) and CPEB2 knockdown (siCPEB2) neurons were detected at a size of around 100 and 135 kDa (see also Supplementary Figure S1 for antibody specificity). (B) Two alternatively spliced forms of CPEB2, rCPEB2a, and rCPEB2b were identified from rat hippocampal neuron cDNA that encoded proteins with additional amino acids (a.a.) at the N-terminus compared with the original mouse CPEB2 clone (NP_787951). The light- and dark-gray boxes indicate unique regions in rCPEB2a and rCPEB2b, respectively. RBD, RNA-binding domain; RRM, RNA recognition motif; Zif, zinc finger. The areas used for antibody production and siRNA knockdown are underlined. (C) CPEB2 expression in siCtrl, siCPEB2, untransfected (mock), and overexpressed (myc–CPEB2a+2b) Neuro-2a cells. The amount of proteins loaded from untransfected and overexpressed cells was 1/50th of that from siCtrl and siCPEB2 cells. (D) Genomic organization of three CPEB2 isoforms. The asterisk denotes the originally reported start codon in the NP_787951 clone. (E) Tissue distribution of CPEB2 in the western blot. The eEF2 signal served as a loading control. Figure source data can be found in Supplementary data. Source Data for Figure 1 [embj2011448-sup-0001.pdf] Download figure Download PowerPoint CPEB2 interacts with eEF2 To understand how CPEB2 regulates translation, the N-terminal 456 a.a. of CPEB2a was used as the bait for a yeast two-hybrid screen to identify its binding partners. The plasmid DNAs isolated from clones of positive interaction were sequenced and listed (Supplementary Table I). Among them, a clone containing a.a. 717–803 of eEF2 was identified (Figure 2A). Additional analysis delineated that the domain V of eEF2 was sufficient to bind to the N-termini of rCPEB2a, rCPEB2b, and mCPEB2 (Figure 2B), suggesting the eEF2-interacting motif is located in the common region of each isoform. The CPEB2–eEF2 association was also confirmed by IP using 293T cell lysates expressing myc–CPEB2a or myc–CPEB2b along with flag–eEF2. Both isoforms pulled down flag–eEF2 (Figure 2C). When eEF2 was divided into half, flag–eEF2N (domains I, G′, and II) and flag–eEF2C (domains III, IV, and V), only the domain V-containing C-terminus was associated with myc–CPEB2a (Figure 2D). Analysis of endogenous CPEB2–eEF2 interaction was performed using Neuro-2a cell lysates. Reciprocal IP showed that CPEB2 co-precipitated with eEF2 and vice versa. This interaction was RNA independent because it was not affected by a treatment with RNase A (Figure 2E). Figure 2.CPEB2 interacts with eEF2. (A) Using the N-terminal 456 a.a. of CPEB2a as the bait, a yeast two-hybrid (Y2H) screen identified a clone containing a.a. 717–803 of eEF2. The various truncated eEF2 mutants were tested for positive (+) or negative (−) association with the CPEB2a N-terminus. (B) The N-termini of CPEB2a, CPEB2b (486 a.a.), and the common region (269 a.a.) were tested for their interaction with domain V of eEF2 in the Y2H system. (C) Co-immunoprecipitation assay. Using 293T cells expressing flag–eEF2 along with myc–CPEB2a or myc–CPEB2b, cell lysates were precipitated with myc antibody (Ab) and immunoblotted with flag Ab. IP: immunoprecipitation, IB: immunoblotting. (D) Using 293T cells expressing flag–eEF2N (domains I, G′, and II) or flag–eEF2C (domains III, IV, and V) along with myc–CPEB2a, cell lysates were precipitated with flag Ab and immunoblotted with myc Ab. (E) Reciprocal immunoprecipitation. Neuro-2a cell lysates, with or without RNase A treatment, were precipitated with control, CPEB2, or eEF2 IgG, and immunoblotted with CPEB2 and eEF2 antibodies. Figure source data can be found in Supplementary data. Source Data for Figure 2 [embj2011448-sup-0002.pdf] Download figure Download PowerPoint CPEB2 inhibits eEF2/ribosome-activated GTP hydrolysis in vitro When complexed with GTP, eEF2 binds to the 80S ribosome that consequently activates its GTPase activity and then catalyses the translocation of the peptidyl-tRNA, deacylated tRNA, and mRNA to allow the next codon to be translated in the decoding site of the ribosome (Merrick and Nyborg, 2000; Herbert and Proud, 2007). Since eEF2/ribosome-triggered GTP hydrolysis is a prerequisite for peptide synthesis, we examined whether this activity was affected by CPEB2. The tissue-isolated eEF2 and 80S ribosome were reconstituted in vitro to monitor GTP hydrolysis (Iwasaki and Kaziro, 1979). Coomassie blue staining of the purified proteins on SDS–PAGE and sucrose density gradient analysis of the 80S ribosome were performed to ensure the quality of preparations (Supplementary Figure S2A). The GTPase activity of eEF2 was upregulated about 20-fold by ribosomes (Supplementary Figure S2B), which was similar to the previous study (Iwasaki and Kaziro, 1979). The rate of eEF2/ribosome-activated GTP hydrolysis was reduced about two-fold by the presence of recombinant (His)6-sumo–CPEB2a compared with the control, enhanced green fluorescent protein fused to Ms2 coat protein ((His)6-sumo—EGFP–Ms2CP), an artificial RNA-binding protein that was used later in the tethered function assay (Figure 3A). CPEB2 partially, but not completely, inhibited GTP hydrolysis even after prolonged incubation (Figure 3A) or when present at 20-fold greater concentrations than eEF2 in the reaction (Supplementary Figure S2C). The CPEB2–eEF2 interaction might preclude eEF2 docking to ribosomes or interfere with the ribosome-induced conformational change of eEF2 to affect GTP hydrolysis (Figure 3B, illustration on the left). To distinguish between the two scenarios, CPEB2 and ribosome mixtures were immunoprecipitated with CPEB2 antibody. Both 28S and 18S ribosomes co-precipitated with CPEB2 only in the presence of eEF2 as judged by the pull down of ribosomal RNAs (Figure 3B), suggesting that CPEB2 bound to ribosomal eEF2 and was likely to be polysome-associated in vivo. Neuro-2a cell lysates treated with or without EDTA were separated on sucrose density gradients. The gradient profiles showed that a portion of CPEB2 protein co-sedimented with polysomes. Because EDTA-induced polysome disassembly resulted in the migration of CPEB2 like the ribosomal protein S6 (RPS6) towards lighter density fractions, CPEB2 was indeed polysome-associated (Figure 3C). Figure 3.CPEB2 decreases eEF2/ribosome-activated GTP hydrolysis in vitro. (A) The rate of ribosome-promoted GTP hydrolysis of eEF2 was determined using purified eEF2 and the 80S ribosome in the absence (none) or presence of recombinant (His)6-sumo-tagged CPEB2a or a control, EGFP–Ms2CP (see also Supplementary Figure S2). Error bars indicate s.e.m. (n=3). One and two asterisks denote significant differences in the amount of hydrolysed GTP at each time point between CPEB2 and EGFP–Ms2CP, with P<0.05 and P<0.01, respectively (Student's t-test). (B) CPEB2–eEF2 interaction may or may not prevent eEF2 from docking to ribosomes, as illustrated. The reactions containing different combinations of CPEB2a, 80S ribosome, and/or eEF2 were precipitated with CPEB2 Ab and analysed for 28S and 18S ribosomal RNAs (rRNAs) by reverse transcription-PCR (RT–PCR). (C) Polysomal distribution of CPEB2. Neuro-2a lysates were treated with or without 50 mM EDTA before sucrose density gradient centrifugation. The proteins from gradient fractions were immunoblotted with CPEB2 and ribosomal protein S6 (RPS6) antibodies. Figure source data can be found in Supplementary data. Source Data for Figure 3 [embj2011448-sup-0003.pdf] Download figure Download PowerPoint CPEB2 represses target RNA translation at elongation To determine whether CPEB2 restrained protein synthesis at elongation due to its inhibitory effect on eEF2, we used the tethered function assay (Huang and Richter, 2007). The C-terminal RNA-binding domain of CPEB2a or CPEB2b was replaced with the dimeric Ms2CP, which recognizes the unique stem-loop sequence (Ms2). EGFP–Ms2CP served as a control. The firefly luciferase reporter was appended with two Ms2 sites at the 3′-UTR in the sense (Luc) or antisense (LucR) orientation. Because Ms2CP does not bind to the complementary Ms2 sequence, the LucR reporter was used as a non-target control of Ms2CP fusions. The internal ribosomal entry site (IRES) from the cricket paralysis virus (CrPV) was added at the 5′-UTR to derive the eIF-independent reporter, CrPV-Luc (Figure 4A), because the CrPV IRES does not require any eIF for translation (Pestova and Hellen, 2003). As all reporter RNA levels were not influenced by CPEB2 (Figure 4B), any change in the firefly luciferase expression would be due to translation. Both CPEB2aN- and CPEB2bN–Ms2CP repressed translation only when binding to the reporter RNAs because they exerted no effect on LucR reporter expression (Figure 4C). Moreover, CPEB2 downregulated both reporter translations including CrPV IRES-directed synthesis, suggesting this inhibition occurred at post-initiation. Next, we examined whether constrained protein synthesis with low doses of inhibitors, specifically at elongation by cycloheximide (CHX) or at initiation by 4EGI-1 (Moerke et al, 2007; Lakkaraju et al, 2008), differentially affected CPEB2-suppressed translation. Since the synthetic molecule 4EGI-1 binds eIF4E and interrupts eIF4E–eIF4G association (Moerke et al, 2007), only the cap- (Luc) but not CrPV-dependent (CrPV-Luc) translation would be affected (Figure 4D). In the presence of CHX but not 4EGI-1, the inhibitory effect of CPEB2 was no longer evident (Figure 4D), supporting that CPEB2 repressed translation at elongation. Intriguingly, despite the rate-limiting step in translation for most RNAs is at initiation (Mathews et al, 2007), CPEB2 suppressed cap-dependent Luc RNA translation even after the initiation capacity was further downregulated by 4EGI-1 (Figure 4D). This indicates that the rate-limiting step for CPEB2-targeted translation takes place at elongation. Figure 4.CPEB2 represses target RNA translation at elongation. (A) The reporter constructs used in the tethered function assay. The firefly luciferase was appended with 3′-UTR containing two Ms2CP-binding sites in sense (Luc) or antisense (LucR) orientation. CrPV-Luc reporter contains hairpin (hp) and internal ribosomal entry site (IRES) sequence in the 5′-UTR. Renilla luciferase was used to normalize variation in transfection efficiency. (B) The RNA-binding domain of CPEB2a (or CPEB2b) was replaced with the dimeric Ms2 coat protein (CPEB2aN–Ms2CP) and EGFP–Ms2CP was used as a control. The 293T cells transfected with the reporters and Ms2CP fusions were analysed for luciferase RNA levels by quantitative RT–PCR or (C) protein levels by dual luciferase assay (normalized: firefly/Renilla). (D) Similarly to (C), except transfected cells were treated with 2 μM cycloheximide (CHX) or 100 μM 4EGI-1 for 12 h before the assay. Error bars indicate s.e.m. (B, D) n=3; (C) n=5. One and two asterisks denote significant differences when compared with the EGFP–Ms2CP control, P<0.05 and P<0.01, respectively (Student's t-test). Download figure Download PowerPoint Translational repression activity of CPEB2 is dependent on binding to eEF2 To define the translational repression motif in CPEB2, various truncated mutants in the N-terminus of CPEB2a were fused to Ms2CP for the tethered function assay (Figure 5A). Amino-terminal truncation mutants of CPEB2a with deletions up to a.a. 187 retained most repression activity, while deletions up to a.a. 364 lost repression activity; therefore, the repression motif is located in the common region of CPEB2 isoforms. Amino acids 381–457, at the end of the CPEB2 N-terminus, were dispensable for repression activity (Figure 5B). Additional mutants with deletions within a.a. 188–364, Δ188–364, Δ188–256, and Δ257–364, showed that the entire region participated in translational repression (Figure 5B). It was recently shown that all four CPEBs shuttle between nucleocytoplasmic compartments, with longer retention time in the cytoplasm (Kan et al, 2010; Lin et al, 2010; Peng et al, 2010). To ensure the lack of repression was not caused by altered distribution of mutant proteins, cells transfected with those constructs were stained with myc antibody to confirm the cytoplasm-prevalent distribution (Supplementary Figure S3). Figure 5.CPEB2-inhibited translation requires an association with eEF2. (A) Schemes of the various truncated myc–CPEB2aN–Ms2CP constructs (see also Supplementary Figure S3 for subcellular distribution of these mutants). (B) The 293T cells transfected with luciferase reporters and Ms2CP fusions were analysed by the luciferase assay (normalized: firefly/Renilla). Error bars indicate s.e.m. (n=3). Two asterisks denote a significant difference, P<0.01 (Student's t-test). (C) The 293T lysates expressing EGFP–Ms2CP or various myc-tagged CPEB2aN–Ms2CP mutants were immunoprecipitated with myc Ab and probed with eEF2 and myc Abs. Figure source data can be found in Supplementary data. Source Data for Figure 5 [embj2011448-sup-0004.pdf] Download figure Download PowerPoint If CPEB2-downregulated translation is mediated through eEF2, the repression activity of CPEB2 mutants should correlate with their eEF2-binding ability. The 293T cell lysates containing CPEB2 mutants with or without repression activity were immunoprecipitated to determine whether they bound to eEF2. The repression-proficient mutants, Δ1–187, Δ381–457, Δ188–256, and Δ257–364, bound to eEF2; whereas the repression-defective mutants, Δ1–256, Δ1–364, and Δ188–364, did not (Figure 5C). When using the eEF2-interacting motif (a.a. 188–364 of CPEB2) to blast the protein database, only CPEB3 and CPEB4 but not CPEB1 were identified (Supplementary Figure S4A), suggesting CPEB3 and CPEB4 may use similar mechanism to control translation. To investigate whether the other repressor, CPEB3, also inhibits translation elongation, the tethered function assay was employed. Not only CPEB3N–Ms2CP repressed CrPV-directed translation (Supplementary Figure S4B) but also the Δ1–364 CPEB3 mutant, which lost its repression activity, was unable to associate with eEF2 (Supplementary Figure S4C–E). The deletion mutant analysis has mapped the a.a. 216–317 is essential for CPEB3 to associate with eEF2, which shares certain degree of sequence similarity with CPEB2's eEF2-interacting motif (highlighted in bold in Supplementary Figure S4A). CPEB2 –eEF2 interaction controls the rate-limiting step of HIF-1α RNA translation at elongation Expression of the short form of CPEB2 (521 a.a.) inhibited translation of HIF-1α RNA as well as a reporter RNA appended with HIF-1α 3′-UTR (Hagele et al, 2009). Several studies have found that a significant portion of HIF-1α RNA is polysome-associated under normoxia (Hui et al, 2006; Galban et al, 2008). However, it is not clear whether CPEB2-hindered HIF-1α synthesis occurs at elongation and thus secure a portion of HIF-1α RNA being polysome-associated. To detect HIF-1α in HeLa cells, the proteasome inhibitor MG132 was used to block degradation. The accumulation of HIF-1α was more sensitive to low concentrations of CHX than high concentrations of 4EGI-1 when compared with c-Myc, supporting that the rate-limiting step of HIF-1α RNA translation is at elongation (Figure 6A). The cells were then transfected with a plasmid expressing full-length (myc–CPEB2a) or eEF2-interacting mutants, the RNA-binding domain (myc–CPEB2C) or myc–CPEB2a Δ188–364, to monitor HIF-1α expression in the presence of MG132 (Figure 6B). The interaction of eEF2 with these CPEB2a mutants and CPEB2b was determined by co-IP (Supplementary Figure S5A). The lack of change in HIF-1α RNA levels showed that expression of CPEB2a, but not CPEB2a mutants, translationally downregulated HIF-1α synthesis (Figure 6C). This was also the case when CPEB2b was overexpressed (Supplementary Figure S5B and C). Overexpression of myc–CPEB2a did not affect global translation as judged by the polysome profiles (Figure 6D, left graphs). Interestingly, the repression of HIF-1α synthesis by wild-type, but not by mutant CPEB2 was accompanied by polysomal accumulation of HIF-1α RNA, showing that CPEB2–eEF2 interaction is required to constrain the rate-limiting step of HIF-1α translation at elongation (Figure 6D). The distribution of control GAPDH RNA was not affected by CPEB2 (Figure 6D). Figure 6.CPEB2 downregulates HIF-1α RNA translation. (A) Western blot analysis of HIF-1α–c-Myc, and actin using lysates from HeLa cells treated with ±20 μM of MG132 and the indicated concentrations of CHX or 4EGI-1. The signals of HIF-1α and c-Myc were quantified and displayed as relative ratios (fold). (B) HeLa cells overexpressing myc–CPEB2a or its eEF2 binding-defective mutants (myc–CPEB2C and Δ188–364) were treated with 20 μM MG132 for 4 h and then used for western blotting (see also Supplementary Figure S5 for the interaction with eEF2), or (C) RNA isolation for quantitative RT–PCR (normalized with the GAPDH RNA level). (D) Two representative polysome profiles from HeLa cells with or without myc–CPEB2a expression. The polysomal distribution of HIF-1α and GAPDH RNAs in HeLa cells expressing myc, myc–CPEB2a, myc–CPEB2C, or the Δ188–364 mutant was determined by quantitative RT–PCR using RNAs isolated from each fraction. (E) HeLa cells transfected with plasmids encoding the two EGFP reporters with or without the HIF-1α 3′-UTR along with myc or myc–CPEB2a plasmid were metabolically labelled with AHA to tag de-novo synthesized polypeptides. EGFP and EGFP–Ms2CP from total cell lysate and the streptavidin-precipitated AHA-labelled proteins were detected by western blotting using EGFP Ab. The newly translated EGFP signals were quantified, expressed as a relative ratio and plotted against the time. (F) The polysome profiles of HeLa cells with or without myc–CPEB2a expression and ±200 μM 4EGI-1 treatment. The polysomal distribution of HIF-1α and GAPDH RNA was determined by quantitative RT–PCR. (G) The amounts of HIF-1α and GAPDH RNAs in the heavy density polysome fractions (#8–11) in (F) were summed and plotted against the treatment time of 4EGI-1. The levels of HIF-1α and GAPDH RNAs at time zero were arbitrarily set to 1. Figure source data can be found in Supplementary data. Source Data for Figure 6 [embj2011448-sup-0005.pdf] Download figure Download PowerPoint To monitor whether the rate of de-novo HIF-1α synthesis is affected by CPEB2, HeLa cells wi
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