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Volume 270, Number 18, Issue of May 5, pp. 10559-10565, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Translational Repression Mediated by the Platelet-derived Growth Factor 2/c- sis mRNA Leader Is Relieved during Megakaryocytic Differentiation (*)

Jeanne Bernstein , Irit Shefler , Orna Elroy-Stein (§)

From the (1) Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Expression of the platelet-derived growth factor 2/c- sis gene is highly restricted and controlled at multiple levels. Its structured mRNA leader, which is unusually long (1022 nucleotides), serves as a potent translational inhibitor. One of the sites of PDGF2 synthesis is megakaryocytes, implying that PDGF2 translation efficiency is modulated during megakaryocytic differentiation. To study the role of the mRNA leader as a translational cis-modulator, the hybrid T7/vaccinia cytoplasmic expression system was used to disconnect between determinants controlling transcription, alternative splicing, and mRNA stability from those controlling translation. Chimeric transcripts in which the human PDGF2/c- sis mRNA leader positioned in frame upstream of a reporter gene were used to determine whether the mRNA leader can confer variable translational efficiencies during differentiation. It is demonstrated that there is a time window during megakaryocytic differentiation of K562 cells in which the strong translational inhibition by PDGF2/c- sis mRNA leader is relieved. The time course of the translational repression relief is similar to that of PDGF2/c- sis transcriptional induction during the differentiation process. A 179-nucleotides CG-rich fragment immediately upstream of the initiator AUG codon is necessary for coffering stringent modulation of the translational efficiency. In NIH3T3 overexpressing translation initiation factor eIF4E, the inhibitory effect of the mRNA leader of c- sis is not relieved, suggesting that the changes in the translational machinery during megakaryocytic differentiation are beyond eIF4E activity. The possible involvement of a 5`-end-independent translational mechanism is discussed.

INTRODUCTION

Platelet-derived growth factor (PDGF)() is known to be involved in the regulation of cell proliferation. This growth factor is a potent mitogen of all cells of mesenchymal origin and has a major role in wound healing and in cellular transformation. It is a homo- or heterodimer of two related polypeptides, one of which, PDGF2, has been shown to be the proto-oncogene c- sis. Expression of PDGF is highly restricted. PDGF2/c- sis can normally be found in blood platelets, activated monocytes, endothelial cells, and placental trophoblasts. However, a large fraction of tumor tissues as well as sarcoma and glioblastoma cell lines express the c- sis mRNA (for review, see Refs. 1-3). PDGF2/c- sis obtained from purified platelets is believed to be synthesized within bone marrow megakaryocytes, the platelet progenitor cells. PDGF2/c- sis is equipped with regulatory elements controlling its transcription and RNA stability (4, 5, 6, 7, 8) . In addition, one of the striking features of c- sis gene is its unusual 5`-untranslated region (5`-UTR) that is composed of 1022 nucleotides and is highly conserved in evolution (9, 10, 11, 12) . PDGF2/c- sis 5`-UTR functions as a potent translational inhibitor (5, 13, 14) , apparently because it imposes a barrier to ribosomal scanning.

Numerous eukaryotic cis-elements controlling transcription have been thoroughly studied. However, very little is known about the contribution of specific cis-elements to the translational control component of gene expression. It is accepted that genes encoding cell growth regulatory proteins are designed to be expressed at low levels to prevent harmful consequences due to their overexpression. This is achieved by transcription and post-transcriptional control mechanisms, one of which is inefficient initiation of translation. It is known that the efficiency of translation initiation is influenced by the sequence context in the vicinity of the AUG codon, the number of upstream AUG codons, and the length and secondary structure of the mRNA leader (for review, see Refs. 15 and 16). Leader sequences of most vertebrate mRNA fall in the size range of 20-100 nucleotides, and an upstream AUG codon occurs in fewer than 10% of the mRNAs (17) . Interestingly, genes for growth factors, cytokine receptors, and proto-oncogenes often produce transcripts with unusually long, structured, upstream AUG-burdened mRNA leaders. Such 5`-UTRs impose a major difficulty to the conventional ribosomal scanning (18) . Coinciding with other mRNAs that encode critical regulatory proteins, the exceptionally long 5`-UTR of c- sis mRNA has three upstream mini open reading frames and high G+C content predicted by computer analysis to form stable secondary structures. Such structures have a calculated free energy that ranges from -100 to -250 kcal mol(9, 14) , capable of preventing the migration of 40 S ribosomes (19) . The three mini cistrons within the 5`-UTR were found to have no regulatory effect on translation (13, 14) .

According to the scanning model for translational initiation, the translation machinery binds to the cap structure at the 5`-end of the mRNA followed by a one-dimensional, ATP-dependent migration along the leader to find a start codon. The initial binding is mediated through eukaryotic initiation factor 4E (eIF4E), which is a subunit of eIF4F complex that functions to unwind secondary structure in the mRNA leader to facilitate ribosomal scanning (20) . Phosphorylated eIF4E is believed to be the rate-limiting component of eIF4F necessary for mRNA association with the 48 S initiation complex (21) . Increased translation levels have been directly correlated with increased phosphorylation levels of eIF4E in response to a variety of growth stimuli (Refs. 22-24 and references therein). As eIF4E overexpression leads to cellular transformation of NIH3T3 cells (25) , a model was proposed predicting that overexpression of active eIF4E can facilitate translation of cellular mRNAs with lengthy and structured 5`-UTRs. Many of such genes, including PDGF2/c- sis, encode for cell growth regulatory proteins, some of which are known proto-oncogenes. However, during cell growth or differentiation, it is hard to determine whether enhanced expression of a specific gene is a result of translation perse that becomes more efficient, since various process such as transcriptional stimulation and mRNA stabilization take place as well.

The fundamental question we wished to address in this study was whether the encumbered 5`-UTR serves invariably as a translational inhibitor or alternatively whether it ``senses'' cellular changes and thus serves as a translational modulator. The later predicts that certain transient changes in the cellular milieu may create supportive conditions for translation of mRNAs that are poorly translated otherwise. Such mechanism might allow for a more efficient translation of c- sis in the right location and timing, i.e. during megakaryocytic differentiation when PDGF is synthesized and stored in -granules prior to the final platelets maturation. A system in which c- sis expression can be induced makes it possible to determine the contribution of different transcriptional, post-transcriptional, and translational regulatory mechanisms during induction. The human hematopoietic stem cell line K562 was chosen for this study since phorbol esters induce its differentiation toward the megakaryocytic lineage with a concomitant induction of c- sis transcription (26) . Since the 5`-UTR of c- sis is known to be a strong translational inhibitor, our working hypothesis was that during the megakaryocytic differentiation process when PDGF2 protein synthesis occurs, an ability to alleviate such inhibition is induced. In this study we have been able to show that there is a time window during megakaryocytic differentiation of K562 cells upon treatment with 12- O-tetradecanoylphorbol-13-acetate (TPA), in which the strong translational inhibitory effect of the lengthy structured 5`-UTR of c- sis is relieved. During the differentiation process, we observed simultaneous induction of c- sis transcription and relief of the 5`-UTR-mediated translational repression. In NIH3T3 overexpressing translation initiation factor eIF4E, the inhibitory effect of the mRNA leader of c- sis was not relieved. This finding suggests that the changes in the translational machinery during megakaryocytic differentiation are beyond eIF4E activity.

EXPERIMENTAL PROCEDURES

Plasmids Constructions

Plasmid pOS14 (27) contains the Escherichia coli chloramphenicol acetyltransferase (CAT) gene placed between the bacteriophage T7 promoter and transcription terminator. The unique NcoI site of pOS14 is located downstream to the T7 promoter and harbors the translational initiation codon ATG of the CAT coding sequence. Plasmids pPD1 and pPD8 were constructed by inserting NcoI fragments of 1022 or 843 bp containing the complete or truncated PDGF2 5`-UTR, respectively, into the NcoI site of pOS14 resulting in placement of the 5`-UTR segments between T7 promoter and the CAT gene. Both 5`-UTR fragments were generated by polymerase chain reaction using pc-sis4.0 (14) as template and the oligonucleotide primer 5`-CCCCCCATGGCGACTCCGGGCCCGGCCC-3`, which is homologous to the most 3` end as well as to a sequence present 179 nucleotides upstream to the 3` end of c- sis 5`-UTR and 5`-CCCCCCATGGTGGCAACTTCTCCTCCTGCA-3`, which is homologous to the most 5` end of the 5`-UTR. pPD9 was constructed by inserting klenow-treated, blunt-ended 1.0-kilobase pair MluI- PvuII fragment of pc-sis4.0 into the Klenow filled-in NcoI site of pOS14.

Cell Culture and Megakaryocytic Differentiation

The chronic myelogenous leukemia cell line K562 (28) was grown in RPMI 1640 supplemented with 50 units/ml of penicillin, 50 µg/ml of streptomycin, and 10% fetal calf serum (Biological Industries, Israel). Megakaryocytic differentiation was induced by treatment of exponentially growing K562 cells with TPA (Sigma) at a concentration of 5 nM. 3T3-pMV7 control cells and 3T3-pMV7-4E cells which overexpress eIF4E, were generous gifts from Dr. N. Sonenberg (McGill University, Montreal, Quebec, Canada) and were grown as described (25) .

Virus Infections and Plasmid Transfections

Transfections were carried out in a 6-well dish using 5 µg of supercoiled plasmid DNA/well. In each well, a total of 9 10 3T3-pMV7 or 3T3-pMV7-4E, 1.7 10 NIH3T3, or 10 control K562 or TPA-treated K562 cells were used. 30 min prior to transfection, the cells were infected with recombinant vaccinia virus vTF7-3 (29) at multiplicity of infection of 1 plaque-forming unit/cell followed by liposome-mediated transfection as described previously (30) . The purified vTF7-3 viral stock was prepared as described (30) .

RNA Analysis and CAT Assay

CAT activity and Northern blot assays for RNA were performed simultaneously on cells infected with recombinant vaccinia vTF7-3 and transfected with the various CAT-expressing plasmids. At 24 h following infection/transfection, the cells were harvested for RNA preparation or CAT activity assay. Total RNA was extracted by the guanidine/phenol extraction procedure (RNAzol reagent, Biotex Laboratories, Inc.). Poly(A) RNA was isolated from noninfected K562 cells using the guanidine/phenol extraction procedure as above followed by purification using oligo(dT) magnetic beads (Dynatech, Inc.). Total or poly(A) RNAs were subjected to Northern blot analysis that was carried out according to standard procedures (31) using P-labeled cDNA probes specific for the CAT gene (32) , c- sis(33) , eIF-4E (25) , -actin (34) , or ribosomal RNA (35) . To quantify the RNA, the intensity of specific bands on the x-ray film was determined by laser densitometry (Ultroscan, LKB), or alternatively, a PhosphorImager (FUJIX BAS100, Fuji) was used. For CAT activity assay, the cell pellet was lysed by three freeze-thaw cycles in 0.1 M Tris, pH 8. CAT activity in the lysates of cells in each well was determined by a phase-extraction assay, which quantitates butyrylated H-labeled chloramphenicol products by liquid scintillation counting following xylene extraction (pCAT Reporter Gene Systems, Promega). Total protein concentration in each sample was determined by the Bradford assay (36) .

Western Blot Analysis

A total of 9 10 control or vaccinia-infected 3T3-pMV7-4E cells were harvested and lysed by 5 min incubation on ice with 50 µl of lysis buffer containing 0.5% Nonidet P-40, 100 mM NaCl, 20 mM Tris-HCl, pH 7.5, 50 mM NaF, and 2 mM phenylmethylsulfonyl fluoride, followed by a 10-min spin in a microcentrifuge. 20 µg of protein of the supernatant as assayed by Bradford (36) was separated by SDS-polyacrylamide gel electrophoresis on a 12% acrylamide gel. Following electrophoresis, the proteins were transferred to a nitrocellulose membrane by semidry blotting using a transfer buffer containing 25 mM Tris-HCl, pH 7.5, 190 mM glycin, and 20% methanol. The membrane was blocked overnight with 5% lowfat dry milk in phosphate-buffered saline at 4 °C followed by a 2-h incubation with rabbit polyclonal antibody against mouse eIF4E and a 1-h incubation with horseradish peroxidase anti rabbit IgG (BioMakor, Israel). All incubations and postincubation washes were done at room temperature using blocking solution. To visualize bands, the membrane was developed using the ECL kit (Amersham Corp.).

RESULTS

The Translational Inhibitory Effect of c-sis 5`-UTR Is Relieved upon Megakaryocytic Differentiation

To study the role of the unusually long c- sis 5`-UTR in mediating translation efficiency during differentiation, it was required to disconnect between determinants controlling transcription, alternative splicing, and mRNA stability from those controlling translation. For this purpose, we have used a vaccinia virus-based expression system. Vaccinia virus, a member of the smallpox virus family, is a cytoplasmic DNA virus encoding for its own transcription and RNA-modifying enzymes. Vaccinia is widely used as a viral expression vector both because of its wide host range (mammalian and avian cells) and because it leads to high expression level of the target gene. The recombinant vaccinia virus vTF7-3 (29) provides expression of the bacteriophage T7 RNA polymerase, a prokaryotic enzyme that does not contain a nuclear localization signal. The prokaryotic RNA polymerase can transcribe any T7 promoter-containing DNA, which is introduced into the cytoplasm of the infected cell. The T7 transcripts are then further modified by the cytoplasmic vaccinia-encoded capping and polyadenylation enzymes and subjected to the host translational machinery. It has been previously shown that the cytoplasmic T7 transcripts generated by the T7/vaccinia system are relatively stable and carry the 5` and 3` T7 promoter- and terminator-derived sequences (37) . Furthermore, it has been shown that differences in expression levels of a target gene represent variations in translational efficiency of the mRNA due to different 5`-UTRs (38) . In the present study, plasmids containing various 5`-UTRs between a reporter gene and T7 promoter were used to transfect TPA-treated or control K562 cells, which have been infected with the vaccinia recombinant virus vTF7-3 that express the cytoplasmic T7 RNA polymerase.

To determine whether differentiation is accompanied by enhanced cellular capacity to translate mRNA harboring certain 5`-UTRs, two plasmids were constructed: pOS14, which produces CAT mRNA with a short leader of 37 nucleotides, and pPD1, which contains the entire 1022 bp of c- sis 5`-UTR joined in-frame with CAT (see Fig. 5a). Differentiation of K562 cells toward the megakaryocytic lineage was induced by treatment with 5 nM TPA for 40 h and was associated with arrest of proliferation, typical morphological changes, and induction of c- sis transcription (not shown). CAT activity as well as CAT mRNA level was determined 24 h following liposome-mediated transfection of each plasmid into vTF7-3-infected K562 cells. Either megakaryocytic-differentiated or control K562 cells were used for the infection/transfection experiments. Fig. 1 a demonstrates the integrity of CAT mRNA made by the vaccinia/T7 cytoplasmic transcription system in both cell types. CAT activity was measured by phase extraction radioassay and normalized against CAT mRNA amount as quantified by laser densitometry. The variations in CAT activity/RNA unit reflect differences in translational efficiencies. To compare the effect of the 5`-UTR in the different cellular environments, the CAT activity/RNA unit of each plasmid in the TPA-treated cells was divided by its value in the control K562 cells. Fig. 1 b demonstrates that the translation efficiency of pOS14-derived CAT was enhanced 1.05 ± 0.07-fold in the TPA-treated compared with the control K562 cells, whereas megakaryocytic differentiation led to 8.46 ± 1.57-fold enhancement of pPD1-derived CAT mRNA translation.

Figure 5: Translation efficiency of CAT mRNA harboring truncated c- sis 5`-UTRs during the course of megakaryocytic differentiation. a, schematic showing of the T7 transcription unit harbored by each plasmid. Lightboxes, c- sis 5`-UTR sequence; darkboxes, the CAT coding sequence; triangle, T7 promoter; circle, T7 terminator. b, log-phase K562 cells were treated with 5 nM TPA for 0-96 h as indicated followed by vTF7-3 infection and transfection of either pOS14, pPD1, pPD9, or pPD8 as described under ``Experimental Procedures.'' 24 h later, CAT activity was determined by the phase extraction radioassay. The presented relative cat activity values are the CAT activity value (cpm/mg of protein) obtained from each plasmid relative to the value obtained from pOS14 at every time point. The bar values represent the average ± S.E. of three to five independent experiments.


Figure 1: Effect of megakaryocytic differentiation on c- sis 5`-UTR-mediated translation. Log phase K562 cells were incubated with 5 nM TPA for 40 h. As described under ``Experimental Procedures,'' the TPA-treated and control K562 cells were infected/transfected with vTF7-3 and pOS14 or pPD1. 24 h following infection/transfection, total RNA was prepared or CAT activity was measured. a, Northern blot analysis of 5 µg of RNA extracted from cells transfected with pOS14 ( lanes1 and 3) or pPD1 ( lanes2 and 4) and probed with P-labeled DNA specific for CAT. Band intensity was measured by laser densitometry. b, CAT activity (cpm/ng of protein) was determined by phase extraction radioassay and divided by the band intensity value of CAT mRNA obtained in each infection/transfection experiment. In each set of transfections, the CAT activity/RNA unit value obtained for each plasmid in the TPA-treated cells was divided by its value in the control K562 cells in order to calculate the effect of megakaryocytic differentiation on the RNA translatability. The bar values represent the average ± S.E. of five independent experiments.


Phorbol esters through protein kinase C can lead to either growth stimulation or differentiation depending on the conditions and cells used. Resting cells exposed to high concentrations of phorbol ester undergo proliferation. Under these conditions, the rapid phosphorylation of several translation initiation factors including eIF4E have been correlated with increase in translation rates (24, 39) . In contrast, exposure of certain log-phase cells to low concentration of phorbol esters results in growth arrest and induction of a long term differentiation process (26, 40) . We wished to assess the effect of the two different TPA treatments on the ability of the cells to translate the pOS14- and pPD1-derived mRNA. NIH3T3 cells, which cannot undergo differentiation but express the receptor for protein kinase C, were used in addition to K562 cells. As shown in Fig. 2 , the translation efficiency of pPD1-derived mRNA was enhanced about 3-fold as a result of a 3-h incubation of either K562 or NIH3T3 cells with 1600 nM TPA. This observation suggests that the immediate events induced by exposure of resting cells to high concentration of TPA might lead to a general phenomenon of enhanced translation of mRNA that bears a cumbersome leader. However, exposure of log-phase cells to the low concentration of TPA (5 nM) normally used to induce differentiation of K562 cells, did not result in a significant relief of the 5`-UTR-mediated translational repression in NIH3T3 cells (Fig. 2 b). This observation suggests that the effect observed in K562 cells (Fig. 1 b) is associated with megakaryocytic differentiation. When K562 cells were treated with hemin to induce their differentiation toward the erythrocytic lineage (41) , such enhanced translation effect was not observed (not shown).

Figure 2: Effect of TPA conditions. A total of 10 K562 cells ( a) or 1.7 10 NIH3T3 cells ( b)/well of a 6-well dish were treated with 5 nM or 1600 nM TPA for 3 or 48 h as indicated. The cells were then infected with vTF7-3 and transfected with pOS14 ( lightbars) or pPD1 ( darkbars). 24 h following infection/transfection, CAT activity was measured as explained under ``Experimental Procedures.'' For each experiment, the CAT activity value of pOS14 in the control untreated cells was arbitrarily set as 1 in order to calculate the effect of c- sis 5`-UTR and of the TPA treatment. The bar values represent the average ± S.E. of three independent experiments.


eIF4E Over-expression Does Not Relieve the Inhibitory Effect of c-sis 5`-UTR

To clarify the role of the rate-limiting initiation factor eIF4E on the translation efficiency of mRNA harboring c- sis 5`-UTR, 3T3-pMV7-4E cells expressing high level of eIF4E described by Lazaris-Karatzas et al.(25) were used. Both 3T3-pMV7-4E and 3T3-pMV7 control cells were infected with vaccinia recombinant vTF7-3 and transfected with either pPD1 or pOS14 followed by analysis of CAT mRNA level and CAT activity 24 h later. To compare the effect of eIF4E overexpression on the translatability of the different 5`-UTR-bearing mRNAs, the CAT activity/RNA unit value obtained for each plasmid in 3T3-4E cells was divided by its value in 3T3-pMV7 cells. Fig. 3 a demonstrates that the translation efficiency of pOS14-derived CAT mRNA was enhanced 2.1 ± 0.24-fold in 3T3-4E compared with 3T3-pMV7 cells, whereas pPD1-derived CAT mRNA activity was enhanced only 1.1 ± 0.04-fold by the eIF4E overexpression. 3T3-pMV7-4E cells did express an elevated amount of recombinant eIF4E as confirmed by Northern blot analysis (not shown). To rule out degradation of eIF4E in 3T3-pMV7-4E cells during the course of the experiment, the cells were infected with vaccinia virus at various multiplicities, and eIF4E level was determined by Western blot analysis at various times after infection. Fig. 3 b shows that eIF4E level in 3T3-pMVA-4E cells remained similar during the 24 h of the experiment, even though a 10-fold excess of virus was used (compare lanes1 and 5). Moreover, it has been reported that during the course of the virus replication, there is neither a significant decrease in the amount of eIF4E nor a significant alteration in eIF4E phosphorylation (42, 43) . According to the observations presented in Fig. 3, it seems that eIF4E by itself is not sufficient for the relief of the c- sis 5`-UTR-mediated translational repression, suggesting that other factors are involved.

Figure 3: Effect of eIF4E. a, 3T3-4E or 3T3-pMV7 cells were infected with vTF7-3 and transfected with pOS14 or pPD1 as described under ``Experimental Procedures.'' 24 h following infection/transfection, total RNA was prepared or CAT activity was determined. The extracted RNA was analyzed by Northern blot analysis probed with P-labeled DNA specific for CAT followed by P-labeled DNA specific for ribosomal RNA. The intensity of CAT- and ribosomal- specific bands was measured by laser densitometry. CAT activity (cpm/ng of protein) was determined by phase extraction radioassay and divided by the band intensity value of CAT/18 S RNA obtained in each infection/transfection experiment. In each set of transfections, the CAT activity/RNA unit value obtained for each plasmid in 3T3-4E cells was divided by its value in 3T3-pMV7 cells in order to calculate the effect of eIF4E overexpression on the RNA translatability. The bar values represent the average ± St.E. of three independent experiments. b, a total of 9 10 NIH3T3-4E cells were infected with vTF7-3 at multiplicity of infection of 1 or 10. At various times postinfection, cells were harvested for detection of eIF4E levels by Western blot analysis as described under ``Experimental Procedures.''


The Relief of c-sis 5`-UTR-mediated Translational Inhibition Parallels the Induction of Its Transcription during the Course of Differentiation

The initial event occurring in response to stimulation with phorbol esters is phosphorylation of key proteins followed by a wide range of cellular consequences at accurate timing. One of the TPA differentiation-induced events in K562 cells is the induction of c- sis transcription (4, 5, 6, 7) . We wished to analyze the time course of both (i) the endogenous c- sis mRNA accumulation and (ii) the translational efficiency of mRNA harboring c- sis 5`-UTR during the differentiation process. To measure the endogenous c- sis mRNA steady state levels, poly(A) mRNA was extracted from K562 cells that had been treated with 5 nM TPA for 0-96 h and analyzed by Northern blot hybridization with a DNA probe specific for c- sis. During the course of differentiation, elevated steady-state c- sis mRNA levels were observed followed by a decline at 96 h after the exposure to TPA (Fig. 4 a). To examine the contribution of the 5`-UTR-mediated translational regulation to the control of c- sis expression during differentiation, the T7/vaccinia hybrid expression system was utilized. CAT was expressed from pOS14 and pPD1 plasmids in K562 cells that had been pre-treated with 5 nM TPA for 0-96 h. It was noted that pOS14-derived CAT activity was hardly changed in the different cellular environment generated throughout the 96 h of the experiment. In sharp contrast, pPD1-derived CAT activity was markedly enhanced for a transient period of time. Fig. 4b represents the CAT activity ratio of pPD1 to pOS14 and thus reflects the time course of the 5`-UTR-mediated translational enhancement during differentiation. The data presented in Fig. 4 suggest that the megakaryocytic differentiation process involves simultaneous induction of both c- sis mRNA accumulation and of the capability to efficiently translate mRNA fused to the inhibitory 5`-UTR of c- sis. This observation suggests that when the cells do not contain any c- sis mRNA they also lack the ability to efficiently translate it.

Figure 4: Time course of c- sis mRNA accumulation and of c- sis 5`-UTR-containing CAT mRNA translation during differentiation. a shows a Northern blot of 4 µg poly(A) RNA from log phase K562 cells that have been treated with 5 nM TPA for 0-96 h as indicated and probed with a P-labeled DNA specific for c- sis followed by P-labeled DNA specific for -actin. RNA bands were quantified by PhosphorImager, and arbitrary units of c- sis/actin mRNA are presented. b, log-phase K562 cells were treated with 5 nM TPA for 0-96 h as indicated followed by infection with vTF7-3 and transfection of pOS14 or pPD1 as explained under ``Experimental Procedures.'' 24 h following infection/transfection, CAT activity was determined by the phase extraction radioassay. The presented relative CAT activity values are the CAT activity value (cpm/mg of protein) obtained from pPD1 plasmid relative to the value obtained from pOS14 plasmid at each time point. The bar values represent the average ± S.E. of five independent experiments.


Effect of Truncations in c-sis 5`-UTR on Translation Efficiency during Differentiation

A highly GC-rich 140-bp sequence immediately preceding the c- sis initiator ATG codon had been shown to be nearly as effective as the entire 5`-UTR in translation inhibition in COS-1 and NIH3T3 cells (14) . This region contains a GC content of 82% implicating a highly stable secondary structure. To examine the role of this sequence in mediating the modulation of translation efficiency during megakaryocytic differentiation, we have constructed plasmids lacking 28 or 179 bp preceding the initiator ATG (pPD9 or pPD8, respectively) (Fig. 5 a). As shown in Fig. 5 b, deletion of 28 bp did not markedly change the translation efficiency pattern conferred by the full-length c- sis 5`-UTR. However, larger deletion, which produced a 843-bp 5`-UTR that lack the GC-rich sequence resulted in less inhibition at zero time and in an extended duration of the enhanced translation phase. The above means that the 5`-UTR with the larger deletion (pPD8) was ``less sensitive'' to cellular changes, suggesting that the CG-rich fragment immediately upstream of the initiator AUG codon is necessary for coffering stringent modulation of translational efficiency during the differentiation process.

DISCUSSION

One of the fascinating aspects of gene expression is the demand for regulation to guarantee correct level, location, and timing. The regulation of c- sis expression is most likely the result of multiple mechanisms. In this study, we have analyzed the effect of c- sis 5`-UTR on translational efficiency during megakaryocytic differentiation of human K562 cells upon TPA treatment. To disconnect between determinants controlling transcription quality/quantity, splicing and RNA stability from those controlling translation, we have employed the prokaryotic bacteriophage T7 transcription machinery in the cytoplasm of K562 cells. The T7/vaccinia is a most efficient transient expression system as 90-100% of the vaccinia-infected cells express the transfected plasmid (44) . The broad host range of vaccinia virus eliminates the technical difficulties of gene transfer into differentiated K562 cells. Such hybrid T7/vaccinia system enabled us to evaluate the 5`-UTR contribution to translation efficiency in different cellular environments, i.e. during megakaryocytic differentiation. In agreement with its performance as inhibitor in COS-1, bovine endothelial cells (14) , and HOS cells (5) , c- sis 5`-UTR operated as a strong translational inhibitor in nondifferentiated K562 cells. However, during the differentiation process, the inhibitory effect of the cumbersome 5`-UTR was strikingly relieved (Fig. 1). This observation shows that the mRNA leader of c- sis is not a translational inhibitor but rather a translational modulator. A 179-bp CG-rich fragment immediately upstream of the initiator AUG codon was found to be necessary for coffering stringent modulation of translation efficiency to heterologous mRNA throughout the differentiation process (Fig. 5).

Among the TPA-triggered cellular changes that eventually lead to differentiation of K562 cells is the induction of c- sis transcription. The accumulation of c- sis mRNA during the course of differentiation has also been observed by others (4, 26, 45) . The present study shows that the 5`-UTR-stimulatory effect on translation of heterologous mRNA has a similar time frame pattern (Fig. 4). It seems that the c- sis gene acquires in parallel both the transcription and translation abilities for a transient period of time during the course of megakaryocytic differentiation. This finding fits a rational of proper timing of events during differentiation, i.e. the translatability of certain mRNA matches the time interval of its maximal abundance. It is unknown whether these phenomena are coupled. However, such a double-safety control mechanism guarantees that no protein will be synthesized from c- sis mRNA in case it is accidentally present in the wrong tissue or at the wrong timing. We have further confirmed that in other cell lines that cannot undergo megakaryocytic differentiation and do not express PDGF such as Hela and J774A, the cis-stimulatory effect on translation is not conferred by c- sis 5`-UTR upon 5 nM TPA treatment (not shown).

One of the global translational control mechanisms is based on the phosphorylation state of the cap binding protein eIF4E. Maximal phosphorylation of eIF4E, reached a few hours following exposure of cells to mitogenic agents, has been correlated with increase in translation rates (22, 23, 24, 39, 46) . It has been suggested that mRNAs harboring structured 5`-UTRs are weak competitors for eIF4F, therefore their translation is dependent upon cellular levels of phosphorylated active eIF4E (25) . This notion was supported by the stimulated translation of the structured 5`-UTR bearing ornithine decarboxylase mRNA upon insulin- or phorbol ester-treatment under conditions leading to eIF4E phosphorylation (39) . The enhanced translation of mRNA harboring synthetic leaders with stable secondary structures at the range of G = -17 to -81.7 kcal mol in NIH3T3 cells overexpressing eIF-4E (47) led to a proposed model to explain the oncogenic effect (25) of eIF4E. The model predicts that the high cellular level of active eIF4E enables the productive translation of proto oncogenes with massive mRNA leaders. Supporting the model, heterologous mRNA harboring the 5`-UTR of ornithine decarboxylase was translated more efficiently in 3T3-pMV7-4E cells, which overexpress eIF4E (48) . In contrast, the translation efficiency of mRNA harboring the structured 5`-UTR of S-adenosylmethionine decarboxylase was not affected by the eIF4E overexpression (48) . Moreover, eIF4E overexpression did not relieve the translational repression of ribosomal protein mRNAs in resting cells (49) . In our hands, eIF4E overexpression did not affect the translation of mRNA harboring the c- sis 5`-UTR that contains secondary structures, of which the stability ranges from G = -100 to -250 kcal mol. However, eIF4E overexpression did enhance the translation of the control mRNA that contains a hairpin 5`-UTR of which the stability is G = -12.4 kcal mol (Fig. 3). More experiments are needed to verify whether eIF4E phosphorylation is required for the rapid increase in the translation of mRNA harboring c- sis 5`-UTR under growth-stimulatory conditions by TPA observed in Fig. 2. In view of the present study, we suggest that during megakaryocytic differentiation of K562 cells eIF4E does not play a key role in the induction of c- sis mRNA translation and that other factors might be involved. It has been recently demonstrated that in contrast to the growth stimulatory effect by mitogens, induction of differentiation is not necessarily associated with enhanced eIF-4E activity. Treatment of log phase HL60 cells with 10 nM phorbol 12-myristate 13-acetate did not result in any substantial shift of eIF4E from free state to heavy subcellular fraction despite a rapid down-regulation of protein synthesis rates.() In addition, differentiation of P19 embryonal carcinoma cells with retinoic acid does not involve phosphorylation of eIF4E.()

The 5`-UTR complexity of a given mRNA determines its translational level in both vertebrate and yeast systems (18, 50) . Specific cis-elements in the mRNAs of ferritin (51) , ribosomal proteins (52) , c- myc(53) , ornithine decarboxylase (39) , transforming growth factor 1 and 3 (54, 55, 56, 57) , proenkephalin (58) , and androgen receptor (59) confer regulated translation on their transcript. Apparently, the expression level of the above genes is regulated at multiple levels. In this study we demonstrate that activation of protein kinase C in K562 cells leads to regulation of PDGF2 expression on both the mRNA steady state and translational levels. In hematopoietic cell lines, protein kinase C activation also exerts distinct levels of regulation on TGF1 expression (54, 60) . We show that the cis-element modulating the translation level of c- sis is within its 5`-UTR. Sequences in the 5`-UTR were shown not to affect the level of c- sis RNA (5) . In addition, translation regulation is not attributes to the minicistrons presence in the 5`-UTR (14) .

It is generally believed (51, 52, 53, 54) that the relative contribution of the cis-elements on translation is dictated by temporal appropriate trans-factors. The present study demonstrates that the 5`-UTR of c- sis confers in cis an ability to respond to modification in the translational machinery during differentiation. Such modifications might facilitate ribosomal scanning from the 5` end of the mRNA by activation of existing translation initiation factors and/or by the induction of RNA helicases and other transacting factors that facilitate the melting of secondary structures. A novel model, distinct from the basic scanning model, describes binding of the translation machinery to an internal site within the mRNA leader independent of its 5` end. Members of picornaviruses that contain exceptionally long, structured, uncapped mRNA leaders utilize the cap-independent mechanism for efficient translation initiation (61) . This alternative mechanism requires high order structures of the 5`-UTR and cellular RNA-binding proteins, some of which have been already identified (62, 63, 64) . It is believed that these proteins mediate ribosomal binding to internal ribosomal entry site within the leader. Interestingly, it has been demonstrated that few cellular mRNAs, which harbor exceptional 5`-UTRs, such as immunoglobulin heavy chain binding protein (65) , fibroblast growth factor-2 (66) , and the homeotic gene Antenopedia (67) , accommodate an internal ribosomal entry site activity. We would like to suggest that temporary appearing transacting factors confer a differentiation-linked internal ribosomal entry site activity to the 5`-UTR of c- sis. This property is anticipated to intensify aspects of expression regulation with regards to timing and cell-type specificity, which might have general implications in cellular growth, differentiation, and development. Our current experiments are designed to address this possibility.

FOOTNOTES

*
This work was supported by grants from the Israel Cancer Research Fund and from the Israeli Health Ministry (to O. E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 972-3-640-9153; Fax: 972-3-642-2046; E-mail: ORNAES@ccsg.tau.ac.il.

The abbreviations used are: PDGF, platelet-derived growth factor; CAT, chloramphenicol acetyltransferase; 5`-UTR, 5` untranslated region; eIF4E, eukaryotic initiation factor 4E; TPA, 12- O-tetradecanoylphorbol-13-acetate; bp, base pair(s).

M. Rao, personal communication.

A. Thomas, personal communication.

ACKNOWLEDGEMENTS

We thank N. Sonenberg for 3T3-pMV7 and 3T3-pMV7-4E cells and for eIF4E probe and antibodies. We also thank I. Fabian for K562 cells, B. Moss for vTF7-3, S. Aaronson for c- sis cDNA, and O. Sella for helpful comments.

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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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