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J. Biol. Chem., Vol. 278, Issue 47, 46357-46368, November 21, 2003
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¶
From the
Departamento de Biologia Celular, Universidade de Brasilia, Brasilia DF 70910-900, Brazil, Embrapa Recursos Genéticos e Biotecnologia, Parque Rural, Final W5, Asa Norte, Brasília DF 70770-900, Brazil, ¶Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Campus da Universidade Federal de Pernambuco, Avenida Moraes Rego s/n, Recife PE 50670-420, Brazil, and the ||Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, United Kingdom
Received for publication, July 15, 2003 , and in revised form, August 25, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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PABP consists of a highly conserved N terminus containing four tandem RNA recognition motifs (RRM) followed by a more variable C terminus (for review, see Ref. 4). The first two RRMs are sufficient for specific poly(A)-binding (11–14). In addition, RRM2 promotes the interaction between PABP and eIF4G (15–17). RRM4 is responsible for most of the nonspecific RNA binding of PABP (12, 13); in yeast the first half of RRM4 is sufficient to confer viability to cells depleted of the normal PABP gene (11). In Xenopus oocytes, RRMs can stimulate translation when tethered to reporter mRNA (18). As to the C terminus, although it does not bind RNA, it enables PABP to multimerize on poly(A) (12). Recently it was shown to include a domain responsible for binding to the PABP-interacting proteins Paip1 and Paip2 as well as to the release factor eRF3 (19). This 74-residue domain (called PABC) is conserved in all PABPs described so far (from protists to vertebrates) and is also present in the hyperplastic discs protein (HYD) family of ubiquitin ligases (20). The structures of both human PABC and its HYD homologue confirmed it as a novel phylogenetically conserved domain responsible for protein-protein interactions (19, 20). The C terminus can contribute to mRNA stabilization (21) and also plays a role in the nuclear export of PABP bound to newly synthesized poly(A)-containing RNA (22).
PABP expression is regulated at the translational level. PABP mRNA is a member of the 5'-terminal oligopyrimidine tract mRNA family (23), which includes mRNAs encoding components of the protein synthesis machinery. Translation of these mRNAs is linked to the growth status of the cell, and this regulation strictly requires a short sequence of polypyrimidines at their 5' end (reviewed in Ref. 24). Control of PABP expression is also achieved through an autoregulatory mechanism whereby PABP binds to an A-rich tract present in the 5'-UTR of its mRNA and represses its own synthesis, when in excess over poly(A)+ RNA (25–28). The A-rich tract, 50–70 nt long, is found in almost all PABP mRNAs (25, 29, 30). In the human mRNA, the 61-nt-long tract contains sets of 5–8 A residues interspersed by 3–6 pyrimidine residues and is located 73 nucleotides from the cap (23). Repression by PABP may be relieved when there is an increase in the intracellular poly(A)+ mRNA, or an increase in the length of pre-existing poly(A) tails. Addition of exogenous poly(A) mimics this effect and specifically stimulates PABP synthesis in vitro in the absence of mRNA synthesis (25), whereas overexpression of PABP in vivo leads to the translational repression of endogenous PABP mRNA (27, 28).
The prototype mechanism for translational repression of mRNAs by 5'-UTR-bound proteins is that described for ferritin mRNA. Regulation of ferritin mRNA translation is mediated by a stem-loop structure called the IRE (iron regulatory element) and its binding factor, the iron regulatory protein (IRP; for reviews, see Refs. 31 and 32). The IRE-IRP complex represses ferritin mRNA translation by precluding the recruitment of the small ribosomal subunit (33). To be effective in vivo and in the rabbit reticulocyte lysate cell-free system, the IRE motif must be located within 60 nucleotides of the mRNA cap structure (34, 35). When placed further from the cap, the IRE-IRP complex delays productive scanning of the ribosomal subunit along the mRNA 5'-UTR, but without greatly compromising overall translation (36). In contrast, in plant or yeast extracts, which lack endogenous IRP, a cap-distal IRE-IRP complex can arrest ribosomal scanning and efficiently inhibit translation (36, 37). As to PABP-mediated translational repression, recent evidence indicates that, in HeLa cells, PABP bound to the A-rich tract in the reporter mRNA 5'-UTR, up to 200 nt from the 5' cap, stalls the migration of the 40 S ribosomal subunit along the mRNA, preventing it from reaching the AUG and forming the elongating 80 S complex (38).
In this study, we examined the requirements of the PABP molecule to act as a translational repressor of reporter mRNAs containing A-tracts in their 5'-UTR. We confirm the lack of positional requirement for effective inhibition by wild type PABP (38). Moreover, we observe that PABP variants lacking the C terminus are much less efficient in repressing the translation of mRNAs carrying cap-distal A-tracts. These PABP variants have affinities for poly(A) similar to that of the full-length protein, but have a reduced ability to associate cooperatively to poly(A). We go on to show a correlation between the presence of the fourth RRM and part of the C terminus, but not including the PABC domain, and the ability of PABP to associate cooperatively to poly(A), multimerize, and enhance PABP-mediated translational repression.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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80 nt) and it lacks a 3' poly(A) tail (we have seen that the presence or absence of such a tail does not affect translation inhibition/stimulation by poly(A)). To reduce the A tract length, this plasmid was digested with StuI and XbaI and the longer fragment, devoid of the original A tract, was ligated to pairs of oligonucleotides designed to reduce the number of A residues to 20 or 10 nucleotides (5' primers: 5'-CCTA20T-3' and 5'-CCTA10T-3'). To reduce the distance between the transcription initiation site and the start of the A tract, the original plasmid was digested with EcoRI and StuI, treated with T4 DNA polymerase, and religated (distance cap/A tract of 10 nucleotides). To increase the distance, the starting plasmid was digested with KpnI and BamHI and ligated with the 62-bp KpnI/BamHI fragment from plasmid Bluescript II KS+ from Stratagene. This plasmid was then digested with XhoI and ClaI, treated with T4 DNA polymerase, and then either religated (distance 86) or ligated with either one or two copies of the 40-nt StuI/SacII fragment obtained from the human PABP cDNA 5'-UTR (distances 126 and 166). All plasmid constructions were confirmed by sequencing.
In Vitro Transcription and Translation—Transcriptions were performed as described previously (25), using T7 RNA polymerase with SalI (cyclin) or BamHI (PABP) linearized plasmid DNAs. For the translation reactions, the rabbit reticulocyte cell-free system (Promega) was treated with micrococcal nuclease to render it mRNA-dependent. In vitro transcribed RNAs were translated at the final concentrations indicated in the figures. Protein synthesis was assayed at 30 °C with [35S]methionine (Amersham Biosciences) and the radiolabeled products analyzed by 15% SDS-polyacrylamide gel electrophoresis and autoradiography. When used, polynucleotides were pre-incubated with the reticulocyte mix for 15 min on ice, prior to addition of mRNA, followed by incubation at 30 °C for 1 h. For the translations with the added proteins, mRNAs were pre-incubated with the GST fusion proteins and ribonuclease inhibitor (final concentration of 200 units/ml in the translation reaction) on ice for 15 min in the presence of Escherichia coli rRNA (2.5 mg/ml) and poly(G) or poly(C) (at 2 mg/ml final) as nonspecific competitors, prior to the addition of the translation extract. All the translation results shown are representative of various experiments performed with different batches of mRNAs and proteins, and the data were highly reproducible in trend from experiment to experiment. Repeating the experiments with the same mRNA/protein batches produced identical results.
GST-PABP Expression Plasmids—The plasmid encoding human PABP (construct P10–636, missing the first nine codons), cloned into the BamHI site of pGEX2T, has been described (25). Clones encoding the PABP variants P10–584, P10–370, and P372–636 were obtained by PCR using an annealing temperature of 50 °C for 30 cycles. As 5' primer for variants P10–584 and P10–370, the oligonucleotide 6291 (5'-CGGGATCCCTGCGGGCAGCCG-3'), which anneals immediately upstream of the PABP translation initiation codon, was used. The 3' oligonucleotides, 3771 and 3772, which are complementary to sequences coding for conserved motifs present at the end of the fourth RRM (3' end of P10–370) and in the second half of the C terminus (3' end of P10–584), have been described previously (39). To clone fragments P10–584 and P10–370 into the pGET2T expression vector, the PCR fragments were digested with NcoI (which cuts on the second methionine at amino acid position 10) and BamHI, prior to in-filling and ligation into the in-filled BamHI-cut pGEX2T vector (Amersham Biosciences). The P372–636 PCR fragment was amplified with the oligonucleotides 7736 (5'-CCGGGATCCGAAGAGCGCCAGGCT-3') and 7737 (5'-CGGAATTCTAGATATTTTTCTTCGGTG-3') and inserted into the BamHI/EcoRI sites of the pGEX2T vector. The P10–370 plasmid was further digested with EcoRI and the larger fragment gel religated to yield the P10–190 plasmid. P237–636 was the result of digesting the plasmid coding for wild type PABP with NcoI/HindIII followed by infilling and religation of the plasmid-containing fragment. P10–542 was obtained by site directed mutagenesis of the P10–636 plasmid to replace the glutamic acid at position 543 with a stop codon. Mutagenesis was performed with the QuikChangeTM site-directed mutagenesis kit (Stratagene). Likewise, P237–542 was obtained from P237–636 by the introduction of the same mutation. All plasmid constructions were confirmed by sequencing. Protein expression and purification has been described previously (25).
Band-shift Assays—The RNA probes used in the band-shift assays were synthesized from linearized plasmids using T7 RNA polymerase and [
-32P]ATP (Amersham Biosciences). Band-shifts were performed as described by Walker et al. (40) with minor modifications. Briefly, 10-µl reactions were carried out in microtiter plates. The labeled probes were initially denatured by incubation at 95 °C for 3 min and then, following rapid cooling, were incubated with different concentrations of the recombinant proteins for 15 min on ice in the presence of binding buffer (5 mM Hepes, pH 7.2, 1.5 mM MgCl2, 2.5% glycerol, and 0.5 mM dithiothreitol), 250 µg/ml competitor E. coli rRNA, 100 mM KCl, 1 mg/ml BSA, and RNase inhibitor (2.5 units). Heparin was then added to 5 mg/ml followed by 2 µl of loading buffer (48.5% glycerol and 0.5% each of bromphenol blue and xylene cyanol). Samples were then immediately run on nondenaturing polyacrylamide gels (acrylamide/bisacrylamide ratio of 60:1) in 0.5x TBE, the gels having been previously pre-run for 15–30 min at 200–300 volts. Electrophoresis was performed at 4 °C, followed by autoradiography.
Pull-down Assays—The glutathione-Sepharose beads (Amersham Biosciences) were initially pre-washed with binding buffer (BB: 150 mM KCl, 5 mM MgCl2, 10 mM Hepes, pH 7.2, 0.2% Nonidet P-40) and saturated with 10 mg/ml BSA in BB for 30 min at 4 °C, followed by two more washes with BB. Approximately 20 µl of the beads were incubated with 4 µg of the GST-PABP variants in a final volume of 100 µl for 30 min at room temperature. The beads were then washed two times with BB containing 4 mg/ml BSA and incubated with 10 µl of translation extract for 30 min at room temperature and washed twice again with BB. Proteins bound to the beads were eluted by addition of SDS-PAGE sample buffer and the samples run on 15% SDS-PAGE, followed by Coomassie Blue staining and autoradiography. Labeled cyclin A and PABP were synthesized in the TNT T7 coupled reticulocyte lysate system (Promega) using 5 µg/ml amounts of each plasmid. The labeled PABP deletions were obtained by linearizations of the wild type PABP cDNA with HindIII (1–237), MscI (1–358), or Tth111I (1–432) (the numbers indicate the amino acids retained from full-length PABP), followed by transcription with T7 RNA polymerase in the presence of the cap analogue and translation in the rabbit reticulocyte lysate. In some cases, prior to their use in the pull-down assays, the reticulocyte lysates were treated with either 100 units/ml cobra venom RNase (RNase V1; Amersham Biosciences) for 30 min at 37 °C or with 250 units/ml micrococcal nuclease for 20 min at 20 °C in the presence of 1.5 mM CaCl2 followed by addition of EGTA to 6 mM. Both RNases efficiently degraded commercial poly(A) (data not shown). When specified, the lysates were also supplemented with E. coli rRNA (250 µg/ml), poly(A) (5 or 20 µg/ml), or poly(C) (20 µg/ml).
Pull-downs with the His-tagged proteins were carried out as described above but using the Ni-NTA resin (Qiagen). Labeled human eIF4G was obtained by subcloning the XbaI/HindIII fragment from plasmid pSK-HFC1 (41) into the same sites of the pBluescript KS vector (Stratagene), followed by linearization with HindIII and in vitro transcription and translation as described above. His-tagged human eIF4A from plasmid pET(His6-eIF4A) (42) and MS2/PABP-(372–636) were expressed and purified on Ni-NTA resin as recommended.
MS2 Tethering Constructs—The reporter plasmids MSC-15 and MSA-15 were obtained by digestion of the MSC-GH and MSA-GH plasmids (43) with XbaI/HindIII and ligation of the larger fragment to the BamHI/HindIII fragment encoding the CAT gene from plasmid pSV2-CAT. Both the vector and insert DNAs were in-filled with Klenow DNA polymerase prior to ligation. The MSC-91 plasmid was constructed by inserting a BamHI/XbaI insert from the polylinker region of plasmid pcDNA3 (Invitrogen) into the BamHI site of MSC-15. In this case, the BamHI ligation was performed first, followed by the in-filling of the protruding ends and a second round of ligation.
To produce the effector plasmids, the DNA fragments encoding the various PABP deletions originally in the pGEX2T vector were first cloned into the pET-MS2 plasmid (21) to generate MS2-PABP fusions. Subcloning the in-filled BamHI PABP fragments from P372–636 and P237–584 into the BamHI site of pET-MS2, also in-filled, yielded MS2/PABP-(372–636) and MS2/PABP-(237–584), respectively. The resulting plasmids were then digested with XbaI/EcoRV, in-filled, and the inserts transferred to the EcoRV site of the eukaryotic expression vector pcDNA3, under the control of the cytomegalovirus promoter. All cloning steps and the final coding frames were confirmed by sequencing.
Transfection Assays—Approximately 4 x 105 human embryonic kidney cells (293-EBNA-BCRJ) were grown in each well of the 24-well dishes with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, streptomycin (100 µg/ml), and penicillin (60 µg/ml). Subconfluent cultures were transfected in DMEM without serum and antibiotics. Transfections were performed with 250 ng of reporter plasmid, 20 ng of pCMV
control plasmid (Clontech), and 2.5, 5, or 10 ng of effector plasmid (for the first two concentrations of the effector plasmid DNA, the difference to 10 ng was supplemented with DNA from the empty pcDNA3 vector). The plasmids were transfected along with LipofectAMINE (Invitrogen) as recommended by the manufacturer, and the transfected cells were allowed to grow for 36 h in DMEM plus 10% fetal calf serum and antibiotics. Prior to harvesting, the cells were washed twice with phosphate-buffered saline. Cells were lysed with lysis buffer (100 mM Tris-HCl, pH 7.8, 0.5% Triton X-100) for 15 min at 37 °C, and the clarified supernatant was assayed for CAT and -galactosidase activities using standard methods (44).
For the RNA expression analysis, 5-fold transfection reactions were carried out using the MSC-91 reporter plasmid with the highest concentration of effector plasmid tested. Approximately 15% of the transfected cells were processed and assayed for CAT and -galactosidase. The remaining transfected cells were extracted with TRIzol (Invitrogen), and the total RNA was digested with RNase-free DNase I (Amersham Biosciences) and then run on denaturing formaldehyde gels prior to Northern blotting. The following DNAs were used as probes: the 0.7-kb XbaI/HindIII fragment of pET-MS2, the 1.6-kb HindIII/BamHI fragment of pSV2-CAT, the 2.4-kb NcoI/SmaI fragment of pGEX2T-PABP, and the 3.5-kb ScaI/NotI fragment of pCMV containing the -galactosidase gene. Probes were labeled with [-32P]dCTP using the Megaprime kit (Amersham Biosciences).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(1–263) PABP, is included in the translation assay (25). Translation of cyclin mRNAs containing in their 5'-UTR either 10, 20 or 61 A residues, at a fixed position relative to the cap (41 nt), was assayed in the absence or presence of increasing amounts of added poly(A) (Fig. 1A). First, we noted that, in the absence of poly(A) competitor, increasing the chain length of A residues progressively reduced the efficiency of translation of cyclin mRNAs, relative to the control mRNA, presumably reflecting enhanced PABP binding. The 10-A-containing cyclin mRNA was not affected by the addition of poly(A), except at the highest doses, which inhibited the translation of the various cyclin reporters and the control RNA. As has been described previously (25, 45), inhibition of translation by poly(A) presumably occurs through a depletion of the lysate PABP, because this inhibition can be reversed by addition of the purified protein (45). The translation of both the 20- and 61-A-containing cyclin mRNAs was stimulated at intermediate poly(A) levels, with similar degrees of stimulation observed for both mRNAs (Fig. 1B). These data are entirely consistent with the finding that PABP can bind oligo(A) sequences as short as 12 nt (11, 12) in vitro. Next, we investigated the importance of the position of the adenylate tract, relative to the 5' end of the mRNA, to the translation of reporter mRNAs. Cyclin RNAs were obtained, which contained 61 A nucleotides placed at various distances from the cap, ranging from 10 to 166 nt. Stimulation of translation by added poly(A) was observed in all five distance reporter mRNAs, although the degree of enhancement was somewhat less efficient in the most cap-distal mRNAs, with A-tracts at 126 and 166 nt from the cap (Fig. 1 (C and D) and data not shown). This difference possibly reflects the presence of additional sequences in the cap-distal mRNAs, or a reduced efficiency of the translation of the mRNAs containing longer UTRs in the reticulocyte lysate. We conclude that PABP-mediated repression in vitro requires more than 10 As in the leader region, and that it can operate even when the A-tract is distal to the cap. This result is in agreement with recently published data (38), as well as with the distance of 73 nucleotides from the cap to the A-tract in the human PABP mRNA (23).
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PABP C Terminus, the Fourth RRM, and Poly(A) Are Required and Sufficient for Specific PABP/PABP Interaction— The experiments above point to the region between amino acids 370–542 as being involved in mediating PABP/PABP interactions and enhancing translational repression. To confirm these results, we used pull-down assays in which PABP variants were bound to glutathione-Sepharose beads and subsequently incubated in the presence or absence of poly(A) with 35S-labeled full-length PABP translated in rabbit reticulocyte lysate. A second 35S-labeled protein, cyclin A, was included in the reaction as a control to monitor for nonspecific bead binding. Following incubation, and washing, the proteins bound to the beads were analyzed by SDS-PAGE, and Coomassie Blue staining and autoradiography. The top panel in Fig. 5A shows that approximately equal amounts of GST-PABP variants were bound and eluted from the beads. As shown in the middle and bottom panels of Fig. 5A, weak and equal cyclin protein binding is observed in all lanes (this nonspecific binding was consistently enhanced following ribonuclease treatment, for unknown reasons). In the presence of poly(A), all N-terminal GST-PABP variants containing at least RRM1/2 pull down 35S-labeled PABP. Comparison with an aliquot of the initial translation mix (Retic lane) allows us to estimate that approximately 20% of the 35S-labeled PABP is bound by GST-PABPs. Variants P237–542 and P237–636, which include a complete RRM4 plus the 370–542 part of the C terminus, identified as necessary for cooperative binding to poly(A) (Fig. 4), or the whole of the C terminus, respectively, also interact with the translated PABP. In contrast, variant P372–636, which only consists of the PABP C terminus, interacts very weakly. This level of binding was well above background binding; as shown in the control lanes, labeled PABP did not bind immobilized GST or glutathione-Sepharose beads. In the absence of poly(A), and with the addition of cobra venom RNase to degrade any remaining excess RNA, most of the fusion proteins, including full-length GST-PABP, lose the ability to interact with 35S-PABP. Surprisingly, the P237–542 and P237–636 variants still retain the capacity to bind to the labeled protein, suggesting that the removal of the first three RRMs from at least one of the binding molecules relieves an inhibitory mechanism, which prevents PABP/PABP binding in the absence of poly(A). The same results were obtained in several independent experiments, some using micrococcal nuclease in place of cobra venom RNase (data not shown).
To rule out the possibility that the P237–636 and P237–542 proteins bind full-length PABP through an RNA bridge (at least when poly(A) is present), we tested their ability to bind 32P-labeled A61 RNA probes using band-shift assays (performed as described in Fig. 4B). The P372–636, P10–636, and P10–370 PABP variants were also included in this experiment as controls (Fig. 5B). Only the proteins containing functional RRMs 1 and 2 bound the A-tract probe, indicating that the P237–636 and P237–542 variants bound full-length PABP directly. We next tested the ability of GST-PABP variants to bind truncated PABP proteins, labeled in vitro, to determine the minimal optimal interaction region. Three different mRNAs, coding for sequential C-terminal PABP deletions, which lack the PABC domain, the whole of the C terminus, and the fourth RRM plus the C terminus, respectively (see Fig. 2A for exact positions of the deletions), were initially co-translated in the rabbit reticulocyte lysate. Following translation, the reaction was treated with micrococcal nuclease, to remove any remaining RNA, and excess ribosomal RNA was added as nonspecific competitor, after the addition of EGTA to quench micrococcal nuclease activity. The reaction was then divided into two aliquots, and either poly(A) or poly(C) was added prior to incubation with various bead-bound GST-PABPs (Fig. 5C). In the presence of poly(A), the three GST-PABPs bring down the three labeled proteins at equivalent levels, compared with their initial reticulocyte levels. Indeed, as observed previously (Fig. 5A), P237–636, although not able to bind poly(A) (Fig. 5B), is as efficient as the P10–370 and P10–636 proteins that do bind poly(A) (Fig. 5C). Even the 1–237 labeled protein that lacks the entire C terminus and the fourth RRM is brought down in similar proportions to the longer proteins. (We also note that the minor labeled proteins, truncated versions of PABP arising from the use of internal AUGs or from premature termination, interact with immobilized PABP.) We cannot rule out a direct interaction between the various proteins but it is more likely that the added poly(A), which allows multiple molecules of PABP to associate, and the endogenous rabbit reticulocyte PABP, act as bridges for any poly(A)-binding protein, resulting in their association with the GST-PABP variants. In the presence of poly(C), as observed above after RNase treatment, neither the wild type nor the C terminus lacking variant (P10–370) can bind any of the labeled protein, whereas P237–636 could relatively efficiently bring down the largest of these proteins (1–432), which retains the first 50 amino acids of the C terminus. With further deletions (protein 1–358, which loses the remaining C terminus), this binding is severely impaired (3-fold reduction in binding), although some minor binding can still be seen even with the labeled protein 1–237 (15% of that observed for protein 1–432). Identical results were obtained with the GST variant P237–542 (data not shown).
Together, these pull-down experiments confirm that the region of the C terminus immediately after the fourth RRM, but excluding the PABC domain, plays a significant role in mediating the interaction between PABP molecules. A role for RRM4 in this self-association is also implied by our findings, because it is required in the GST fusion for maximal interaction (compare P372–636 and P237–542/636; Fig. 5A) and its removal from the labeled PABP deletion abolishes most of the remaining binding activity to the P237–636 variant in the presence of poly(C) (Fig. 5C). Alternatively, the residual activity observed between P237–636 and the labeled protein 1–237 may indicate minor binding to the other RRMs, even in the absence of RRM4. This latter observation is consistent with the overall sequence similarity between RRMs 2, 3, and 4 of 45–50% in human PABP, with RRM1 being more divergent. In summary, gel-shift assays and pull-down experiments indicate that RRM4 and the part of the C terminus, up to but not including PABC, mediates PABP self-association. This interaction is dramatically enhanced in the presence of poly(A); in the absence of poly(A), only truncated versions of PABP, lacking RRM1/2, retain the ability to bind other PABP molecules.
Tethered PABP C Terminus Enhances Translation Repression by MS2 of mRNA Containing MS2 Binding Sites in Their 5'-UTR—To investigate the role of the C terminus in enhancing translation repression by PABP in vivo, we undertook a tethered strategy whereby MS2 binding sites were inserted in the 5'-UTR of CAT reporter genes and, in a second plasmid, the MS2 protein was fused to either the PABP C terminus (amino acids 372–636) or its fourth RRM plus the proximal part of the C terminus (amino acids 237–542 missing the PABC domain), or used alone (Fig. 6A). The MS2 protein is known to repress translation/expression of reporter genes containing MS2 binding sites in their 5'-UTR, as long as it is placed within the first 40 or so nucleotides after the cap. It is not very efficient at preventing translation if the distance between the cap and the MS2 binding site is increased further (43, 47). Fig. 6 (B and C) describes the scheme of the CAT reporters and the MS2 fusion proteins. Two different CAT constructs were made with the high affinity MS2 binding site MSC placed at 15 and 91 nucleotides from the transcription start site, MSC-15 and MSC-91 respectively, and a third construct contained the low affinity MS2 binding site MSA placed at the 15-nucleotide distance, MSA-15 (47).
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-galactosidase to serve as transfection control. After 30–35 h of transfection, the cells were harvested and assayed for CAT and -galactosidase activities. To compensate for variations in transfection efficiency, the ratios between the CAT and -galactosidase activities were determined and for each set of transfections, this ratio was compared with a control transfection where no MS2 expression plasmid was included (Ctrl). In some experiments, RNA was extracted as well and used for Northern blots to confirm approximately equal expression and stability of the different CAT and MS2 genes (Fig. 7D). First, the cells were transfected with the MSC-15 CAT reporter plasmid along with increasing concentrations of the plasmids expressing the different MS2 fusion proteins (Fig. 7A). All three proteins led to a dose-dependent decrease in CAT activity, with a maximum reduction of 40%. A minor enhancement of repression was observed by the fusion proteins containing the PABP C terminus. In cells transfected with the reporters containing the mutated MS2 binding site (MSA-15), no reduction in CAT activity was observed for MS2 on its own and less than 20% reduction for the MS2-PABP C terminus fusions, showing that expression of the MS2 fusions is not causing a general nonspecific inhibition of CAT expression (Fig. 7B). We then analyzed the effect of the fusion proteins on the reporter CAT mRNA bearing a cap-distal MS2 binding site (MSC-91, Fig. 7C). Although less than 10% reduction of CAT activity was observed even with the highest dose of the MS2 plasmid tested, both fusions containing the PABP fragments were still capable of efficiently (40% reduction) repressing CAT expression. These effects were largely at the level of translation, as we did not observe corresponding changes in RNA levels (Fig. 7D). The tethered strategy has also been used by us to specifically investigate reduction in CAT mRNA abundance as well as translation in 293 cells mediated by PABP binding to its 5'-UTR (48). Full-length PABP fused to MS2, like the MS2/C terminus fusions described above, despite reducing the expression of the CAT construct MSC-91, does not affect the reporter mRNA levels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Translation of PABP mRNA is controlled by at least two independent mechanisms: autoregulation mediated by the PABP protein (25–27), and coupling to the cellular growth stage mediated by the polypyrimidine stretch in its 5' end (23). In this study, by using cyclin reporter mRNAs with the A-tracts in their 5'-UTR, we were able to concentrate on one aspect of PABP mRNA translation, avoiding the influence of other sequences present in PABP mRNA, which may affect its translation or stability (28). The A-rich sequence required for PABP mRNA translation repression differs from other sequences that have a similar function such as the IRE, in that it does not easily assume a secondary or tertiary structure, which may prevent ribosomes from scanning along the mRNA. The A-rich sequence also does not need to be positioned proximally to the cap structure (this work and Ref. 38). We reasoned that specific properties of PABP might be required for it to be an effective repressor and decided to use this activity as a tool to study the contribution of individual domains within PABP to its function. Full-length PABP represses the translation of cyclin mRNAs bearing A-tracts in their 5'-UTR, whether the A-tracts are proximal or distal to the cap, although with somewhat higher efficiency in the former case. On the other hand, repression by PABP variants lacking the C terminus is much more sensitive to the distance of the A-tract from the cap (Fig. 3). Thus, the C-terminal region of the PABP, although not interfering with binding to poly(A), is involved in the repression mechanism. Its role may be to increase the size of the complex bound to the RNA, not only by recruiting other PABP molecules but perhaps also by allowing the association of other proteins to create a multiprotein complex that blocks ribosomal scanning. Previous reports also hint at the contribution of the C terminus to PABP function. In Xenopus oocytes, a minimal PABP consisting of RRMs 1 and 2 did not seem to be able to bind efficiently to mRNAs or prevent their deadenylation when overexpressed in oocytes, in contrast to wild type PABP (49). In a HeLa-derived cell line, a similar PABP variant (as well as one containing the four RRMs but no C terminus) was still able to bind efficiently to poly(A) and shuttle to the cellular nucleus although its export back to the cytoplasm was impaired (22).
The C-terminal region of PABP was previously reported to be involved in mediating protein/protein interactions, and more specifically PABP multimerization in the presence of poly(A) (12). The recent description of the PABC domain very much filled a gap in the knowledge about PABP function by mapping the motif involved in interactions with eRF3, PAIP1 and PAIP2 (19, 50, 51). Our results build upon these discoveries by confirming the participation of the C terminus in PABP/PABP interaction, while ruling out any participation of the PABC domain, and suggest a significant additional contribution of the fourth RRM. These results may relate to those obtained in yeast, where neither the first two RRMs nor the last 50 amino acids, which includes most of the PABC domain, are involved in mRNA stabilization by PABP (21). The fourth RRM is one of the least characterized of the PABP RRMs. An analysis of sequence conservation of RRMs from PABPs of distantly related organisms shows that RRM 4 is as conserved as RRM 2, which is known to be involved in such important functions as specific poly(A) binding and interaction with eIF4G. In contrast, RRM 3 is much less conserved. RRM 4 has been ascribed roles in cellular viability and in translation (see Introduction), but no interacting factors have yet been identified. Although its involvement in the protein/protein interactions necessary for PABP multimerization may in part explain its conservation (this work), it is also possible that it interacts with still unknown protein or RNA factors (18).
The region of the C terminus bordered by RRM 4 and the PABC domain first came to notice when PABP sequences from yeast and human were compared, because of its high content of the amino acids proline and glutamine (52–54) (Fig. 8). Strikingly, in human PABP, roughly 1 in every 5 or 6 amino acids in this region is proline and 1 in every 3 is either glutamine, asparagine, or arginine, and there are no negatively charged amino acids in the entire 150-amino acid region. Alignments of the C terminus plus the fourth RRMs from PABPs of distantly related organisms shows no consistent sequence homology within this "linker" region, although the bias for the amino acids proline, glutamine, and, to a lesser extent, asparagine and arginine is maintained, as is the lack of acidic residues (Fig. 8).
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Evidence from various sources indicates that PABP activity can be regulated by different means. An example of such modulation has been observed in mammals, where binding of human eRF3 to PABP abolishes its ability to multimerize when binding to poly(A) (56). Phosphorylation of plant PABP has also been shown to play a role in regulating its binding to poly(A), because only the modified form binds poly(A) cooperatively (57). Theoretically, phosphorylation of the proline linker region could reduce its net positive charge and enhance PABP-PABP interaction. Recently, human PABP was identified as a substrate of the coactivator-associated arginine methyltransferase or CARM1 (58). Modification of target proteins by arginine methyltransferases has been shown to interfere with protein-protein interactions, and, in one example, the arginine residues flank proline-rich motifs (59). Intriguingly, the region in PABP methylated by CARM1 has been mapped to the amino acids 384–478 (58), within the linker region that we show to be involved in PABP-PABP interaction (see Fig. 8). However, because the recombinant protein binds cooperatively to poly(A), at least for human PABP, neither methylation nor phosphorylation is probably required for the PABP-PABP interaction. Nevertheless in vivo, these modifications might play an important role in regulating poly(A)-binding and PABP-PABP interactions. Further work is still needed to elucidate these possibilities and to define precisely how the multiple PABP domains interact.
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FOOTNOTES |
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** To whom correspondence should be addressed. Tel.: 55-81-3301-2568; Fax: 55-81-3453-2449; E-mail: opmn{at}cpqam.fiocruz.br.
1 The abbreviations used are: PABP, poly(A)-binding protein; RRM, RNA recognition motif; CAT, chloramphenicol acetyltransferase; nt, nucleotide(s); UTR, untranslated region; IRE, iron regulatory element; IRP, iron regulatory protein; GST, glutathione S-transferase; BB, binding buffer; BSA, bovine serum albumin; Ni-NTA, nickel-nitrilotriacetic acid; DMEM, Dulbecco's modified Eagle's medium.
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