To whom correspondence should be addressed: Div. of Cardiovascular Research, Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei 11529, Taiwan, ROC. Tel.: 886-2-2652-3931; Fax: 886-2-2782-9143
* This work was supported by Grant NSC-88-2314-B-001-026 from the National Science Council of Taiwan, Republic of China.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We have previously demonstrated that phorbol myristate acetate (PMA) up-regulates H-ferritin gene expression in myeloid cells by stabilization of its message. In the present report, we showed that insertion of the 3′-untranslated region (3′-UTR) of H-ferritin mRNA at the 3′-end of luciferase coding sequence significantly reduced the stability of luciferase mRNA in human monocytic THP-1 cells. However, the half-life of the chimeric transcript was markedly prolonged after PMA treatment. A cytosolic protein factor from THP-1 cells was found to specifically bind to H-ferritin 3′-UTR. PMA treatment of THP-1 cells resulted in the reduction of the RNA binding activity in a time-dependent manner. Deletion analysis and RNase T1 mapping revealed a pyrimidine-rich sequence within the 3′-UTR which interacts with the protein factor. Competition experiments with homoribopolymers further demonstrated the importance of uridines for the binding activity. Point mutations in uridines of the pyrimidine-rich sequence reduced the protein binding to 3′-UTR, while increasing the stability of the chimeric luciferase transcript. Together, these results demonstrate that the pyrimidine-rich sequence in the 3′-UTR is involved in post-transcriptional regulation of H-ferritin gene expression in myeloid cells.
phorbol 12-myristate 13-acetate
reverse transcriptase-polymerase chain reaction
Ferritin is a multimeric protein with a function in controlling the iron homeostasis in cells (
). In mammals, each ferritin molecule consists of 24 subunits of two types, heavy (H)1 and light (L), which are derived from distinct genes and share only about 50% homology in amino acid sequences. It is generally believed that H-ferritin, which contains a ferroxidase activity (
), plays a key role in the intracellular flux of iron, whereas L-ferritin is primarily responsible for the iron storage. H- and L-ferritin combine in variable ratios in different cells or tissues depending on the cellular requirement for iron as well as the differentiation or pathological states. The synthesis of H- and L-ferritin can be regulated by iron at the translational level (
). When the intracellular iron concentration is low, a repressor protein would bind to a conserved iron regulatory element located in the 5′-untranslated regions of H- and L-ferritin mRNAs to inhibit the translation of both genes (
). Increased iron concentration will lead to the dissociation of the repressor from the iron regulatory element and result in the increase of ferritin synthesis. In addition, early studies have demonstrated that L- and H-ferritin genes are subjected to differential regulation during development, cellular differentiation, or inflammation, although the molecular mechanisms are not fully resolved (
). Using the human monocytic THP-1 cell line as a model system, we have demonstrated that phorbol myristate acetate (PMA)-induced differentiation of THP-1 cells toward macrophages markedly up-regulates the expression of H-ferritin mRNA, but not L-ferritin mRNA, in a cell-type specific manner (
), we hypothesized that an unique cis-regulatory element within the 3′-untranslated region (3′-UTR) of H-ferritin mRNA is likely involved in regulating the stability of the H-ferritin message in PMA-treated THP-1 cells.
In the present study, we identified a pyrimidine-rich sequence within the 3′-UTR of the H-ferritin mRNA to be associated with the message turnover in THP-1 cells. Gel-mobility shift assay demonstrated the existence of a RNA-binding protein in the cytosolic fraction of THP-1 cells to interact with this sequence. PMA treatment down-regulated the binding of the protein factor to H-ferritin 3′-UTR, suggesting that the interaction between the cytosolic protein and the cis element serves as a destabilizing signal to facilitate the degradation of H-ferritin mRNA in THP-1 cells.
Human monocytic THP-1 cells were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum. HeLa and hepatoma PLC cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were incubated in a humidified atmosphere of 95% air, 5% CO2 at 37 °C. For PMA treatment, THP-1 cells were grown to a density of 1 × 106 cells/ml and treated with 100 ng/ml PMA for indicated times.
A cDNA fragment spanning the full-length 3′-UTR of the H-ferritin gene (nucleotides −3 to +150, +1 is the first nucleotide after the stop codon) was obtained by polymerase chain reaction (PCR) using the human H-ferritin cDNA as the template (
). EcoRI and HindIII restriction sites were created in sense and antisense primers, respectively. The sequences of the sense and antisense oligonucleotides are as follows: sense, 5′-GACAGTGATAATGAATTCTAAGCCTCGGG-3′; and antisense, 5′-GGGACCAAGCTTCTTTATTTGAAGGAATGG-3′. The PCR fragment was subcloned into the EcoRI-HindIII site of the pGEM11Zf+ vector (Promega). To prepare the constructs containing the mutated nucleotides within the sequence located at nuucleotides +72 to +88 of the 3′-UTR, the sense primers containing mutated nucleotides and theSphI restriction site at the 5′-end were used for PCR. The sequences of these oligonucleotides are as follows: M1, 5′-GGCAGTGCATGCATGTTGGGGATACCTT-3′; M2, 5′-GGCAGTGCATGCATGTTGGGGATACCATAACCATATCTAT-3′. The PCR was performed with M1 or M2 sense primer and the antisense primer containing theHindIII restriction site described above. The PCR fragment was digested with SphI and HindIII restriction enzymes and subcloned into the SphI-HindIII site of digested pGEM11Zf+ plasmid containing the wild type 3′-UTR. To construct the luciferase/3′-UTR chimeric plasmid, a cDNA fragment containing luciferase coding sequence was prepared by PCR using pGL2-Basic plasmid DNA as the template. The sequences of the primers used are as follows: sense, 5′-GATCTAAGTAAGCTTGGCATTCCGGTACTG-3′; antisense, 5′-CGCTGGATCCAGTTACATTTTACAATTTGG-3′. The PCR fragment was subcloned into the HindIII-BamHI site of the mammalian expression vector, pcDNA3, to generate pcDNA3-Luc. The cDNA fragment of wild type or mutant 3′-UTR withXhoI and ApaI restriction sites created at the 5′- and 3′-ends, respectively, was prepared by PCR and subcloned into the XhoI-ApaI site of pcDNA3-Luc to generate wild type or mutant pLuc-HF-3′UTR.
Transient Transfection and RT-PCR
THP-1 cells (2 × 107) were transfected with 2 μg of pcDNA3-Luc or pLuc-HF-3′UTR plasmid DNA using Effectene reagent (Qiagen) according to the manufacturer's instructions. After incubation in culture medium for 24 h, cells were divided into 4 dishes and each dish was treated with 10 μg/ml actinomycin D for indicated times. Total RNA was then isolated and the expression level of luciferase mRNA was quantified by RT-PCR. Briefly, 1 μg of total RNA was reverse transcribed into cDNA by incubation with 200 units of Maloney leukemia transcriptase (Life Technologies, Inc.) in 20 μl of reaction buffer containing 10 units of RNasin, 0.2 μg of random hexamers, and 0.8 mm dNTPs at 37 °C for 1 h. Reaction was terminated by heating at 95 °C for 10 min and the mixture was diluted to 500 μl with deionized H2O. An aliquot (2.5 μl) was taken for PCR amplification with the primers described above for the preparation of luciferase cDNA. The PCR was performed in 25 μl of 10 mm Tris-HCl, pH 8.3, containing 50 mm KCl, 1.5 mm MgCl2, 0.2 mm dNTPs, 1 μm of each primer, and 1.25 units of Taq DNA polymerase. The reaction proceeded for 30 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, and extension at 72 °C for 1 min. Amplified cDNA was electrophoresed on 1% agarose gels containing ethidium bromide. The cDNA was visualized under UV light and the images were analyzed by UVP's Gel Base Windows Software.
Preparation of Cytosolic Extracts and Gel Mobility Shift Assay
Cells were harvested by centrifugation at 200 ×g for 5 min, washed twice with ice-cold phosphate-buffered saline, and centrifuged again. Cell pellets were resuspended in ice-cold hypotonic buffer containing 25 mm Tris-HCl, pH 7.9, 0.1 mm EDTA, 1 mmNa3VO4, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 0.2% Nonidet P-40. After placing on ice for 5 min, cell lysate was subjected to centrifugation at 100,000 × g for 30 min at 4 °C. The supernatant (cytosolic fraction) was removed and stored at −70 °C. The protein concentration was determined by a Bio-Rad protein assay. To prepare the radiolabeled transcript of the H-ferritin 3′-UTR, the pGEM 11Zf+ plasmid containing wild type or mutant 3′-UTR was linearized byHindIII restriction enzyme digestion. In vitrotranscription was performed with T7 polymerase in a reaction containing 0.4 mm unlabeled ATP, GTP, CTP, 0.04 mm UTP, and 30 μCi of [α-32P]UTP (Amersham Pharmacia Biotech, >3000 Ci/mmol). The 32P-labeled transcript was filtered through a TE-100 Chroma spin column (CLONTECH) to remove free [32P]UTP. For the RNA binding reaction, 10–40 μg of cytosolic proteins were incubated with32P-labeled transcript (30,000–50,000 cpm) in 20 μl of 15 mm HEPES, pH 7.4, containing 1 mm EDTA, 50 mm KCl, 3 mm MgCl2, 10% glycerol, and 10 μg/ml yeast tRNA for 30 min at room temperature. Twenty units of RNase T1 were added and incubation continued for an additional 30 min, followed by addition of heparin to a final concentration of 2 mg/ml for an additional 10 min. Samples were then subjected to electrophoresis performed on a 6% native polyacrylamide gel using 0.25 × TBE as electrophoresis buffer. Gels were dried and exposed to Kodak X-Omat AR films overnight at −70 °C.
RNase T1 Mapping
Following electrophoresis of the binding reaction, gel was exposed to x-ray film at 4 °C overnight. The region of specific RNA-protein complex was identified and excised. The protected RNA fragment was eluted from gels by electrophoresis through a NA45 membrane and then recovered from membrane by extraction with high salt buffer. After ethanol precipitation, the recovered RNA was digested with or without RNase T1 (20 units) at 37 °C for 30 min. The 32P-labeled 3′-UTR transcript was also subjected to RNase T1 digestion. The digested products were analyzed by a 12% polyacrylamide, 8 m urea sequencing gel.
Following gel-mobility shift assay, the radiolabeled RNA-protein complex band was excised from the wet gel as described above. The gel slice was placed on ice and irradiated under a 254-nm UV lamp at a distance of 4 cm for 20 min. The gel slice was then treated with 50 μl of 10 mg/ml RNase A solution at 37 °C for 30 min, followed by the addition of 10 μl of 6 × SDS sample buffer. After incubation at 37 °C for an additional 30 min, the entire gel slice was loaded into a 10% SDS-polyacrylamide gel and analyzed by autoradiography.
To explore the possibility that the 3′-UTR of H-ferritin mRNA contains a regulatory element affecting mRNA stability, a chimeric luciferase reporter gene construct carrying the entire H-ferritin 3′-UTR at the 3′-end of the luciferase coding sequence was prepared. After transient transfection into THP-1 cells, the expression of chimeric luciferase mRNA was assessed by semiquantitative RT-PCR. As shown in Fig. 1, insertion of H-ferritin 3′-UTR led to the decrease in the half-life of luciferase mRNA from 8.0 ± 0.5 to 4.5 ± 0.3 h as measured in the presence of actinomycin D. PMA treatment markedly prolonged the half-life of the chimeric transcript to greater than 9 h. In contrast, the half-life of the parental transcript was not significantly altered by PMA treatment. This result supports the idea that the H-ferritin 3′-UTR contains a regulatory sequence mediating the effect of PMA. When the 32P-labeled transcript of H-ferritin 3′-UTR was incubated with the cytosolic extracts prepared from monocytic THP-1 cells, followed by digestion with RNase T1, and analyzed by a native polyacrylamide gel, a RNase-resistant band was observed (Fig. 2). This band was abolished by the addition of proteinase K or SDS in the binding reaction, indicating that it was formed by the interaction of a protein factor with the radiolabeled RNA probe. The RNA binding activity was proportional to the increments of the cytosolic proteins (Fig.3A). When the radiolabeled antisense RNA probe was used, there was virtually no binding activity detected with the same amounts of proteins. The sequence specificity of the RNA- protein complex was further revealed by the competition experiment showing that the complex formation was inhibited by the addition of excess amounts of unlabeled 3′-UTR (Fig. 3C). Further experiments demonstrated that the RNA binding activity was barely detectable in the cytosolic extracts prepared from HeLa or hepatoma PLC cells, indicating that the protein factor binding to the H-ferritin 3′-UTR is predominantly present in myeloid cells (Fig.3B). The effect of PMA treatment on the RNA-protein complex formation was examined. As shown in Fig.4A, the RNA binding activity in PMA-treated THP-1 cells was substantially less than that in control cells. Time course experiments further revealed that down-regulation in RNA binding is evident at 3 h, reaches a maximum at 12 h, and is prolonged to 36 h following PMA treatment (Fig.4B).
To locate the sequence interacting with the cytosolic protein factor, the 3′-deletion RNA probes were prepared and used for the binding assay. As shown in Fig. 5, when the RNA probe prepared from the AflII-digested 3′-UTR cDNA template was used for binding assay, the RNA-protein complex was still observed. This complex, however, was not detected by using shorter RNA probes prepared from the SphI-digested DNA template. This result indicates that the sequence responsible for the interaction with the protein factor is located in the region between SphI andAflII restriction sites in H-ferritin 3′-UTR. This sequence was further identified by RNase T1 mapping assay. As shown in Fig.6, digestion of the entire32P-labeled 3′-UTR with RNase T1, which cleaves after each G residue, results in oligonucleotides varying from 22 through 2 bases. When the protected 32P-labeled RNA fragment isolated from the RNA-protein complex was electrophoresed, it migrated as a 22-base oligonucleotide. Further digestion of the protected RNA fragment with RNase T1 did not yield smaller fragments, indicating that the protected RNA is a G-free sequence located at 72–92 nucleotides downstream of the stop codon of H-ferritin gene. It was noted that this sequence is rich in pyrimidines, particularly the uridines. To test whether the U residues are important for binding with the protein factor, a competition experiment with various homopolymers of ribunucleotides was performed. As shown in Fig. 7, the formation of the RNA-protein complex could be completely inhibited by excess amounts of poly(U) homopolymers but not by poly(C) or poly(A), supporting that the poly(U) tract is essential for the binding activity.
To confirm the role of the U-rich sequence on mRNA stability, the effects of point mutations within the poly(U) tract of the H-ferritin 3′-UTR on the RNA-protein complex formation and the stability of chimeric transcript were examined. As shown in Fig.8B, substitution of uridines at positions +72 and +74 by adenines (M1) reduced the binding of the cytosolic protein factor to 3′-UTR by ∼50%. Further substitution of uridines at positions +77, +79, +83, and +85 by adenines (M2) completely abolished the binding. When the chimeric luciferase constructs carrying the mutant 3′-UTRs were transfected into THP-1 cells and the stability of their transcripts was assessed, it was shown that the mutant Luc/M1 mRNA has a calculated half-life of 7.4 ± 1.4 h, and mutant Luc/M2 mRNA has a half-life of >9 h. Apparently, both mutant transcripts are more stable than the chimeric transcript carrying the wild type 3′-UTR shown in Fig. 1.
As revealed by the transient transfection experiment, the 3′-UTR of the human H-ferritin gene placed at the 3′-end of luciferase gene destabilized the reporter transcript by decreasing its half-life from 8.0 ± 0.5 to 4.5 ± 0.3 h in monocytic THP-1 cells. Nevertheless, PMA treatment significantly prolonged the half-life of the chimeric mRNA to greater than 9 h, indicating that the 3′-UTR contains a sequence determinant mediating the PMA-induced message stabilization in these cells. Identification and sequence analysis of this regulatory element revealed that it is pyrimidine-rich and interacts with a novel protein factor which is present in cytoplasm of THP-1 cells but not HeLa or hepatoma PLC cells. The restriction in cell-type distribution is consistent with the early finding that induction of H-ferritin gene expression by PMA is myeloid cell-specific (
). When we examined the 3′-UTRs of H-ferritin mRNAs from different species, it is clearly shown that the homologous pyrimidine-rich sequence is present in all of them (Fig.9), indicating that this sequence is highly conserved and may have an important role in H-ferritin gene expression. PMA treatment of THP-1 cells resulted in the decrease of the protein binding to the H-ferritin 3′-UTR, suggesting that the binding protein acts as a destabilizer. A similar phenomenon has been reported in 3T3 cells, in which the induction of ribonucleotide reductase R1 and R2 genes by PMA is associated with the decrease in the binding activities of the RNA-binding proteins to their 3′-UTRs (
). Likewise, a recent study on human pleural mesothelioma cells has shown that stabilization of the urokinase receptor mRNA by PMA is correlated with the down-regulation of the formation of a urokinase receptor RNA-protein complex (
The competition experiment with homoribopolymers demonstrated the importance of poly(U) within the pyrimidine-rich sequence for the interaction with the protein factor. The correlation between protein binding to the U-rich sequence and message stability was further supported by the observation that mutations in some of the uridines led to the reduction in RNA binding, but increase in the stability of mutant chimeric luciferase mRNA. Recently, numerous studies have demonstrated the involvement of U-rich regions in 3′-UTRs to regulate the message stability in many mRNAs. Accumulative evidence has revealed that the AUUUA pentamer and U-rich sequence present in 3′-UTRs of unstable mRNAs encoding cytokines, lymphokines, oncogenes, and growth factors plays an important role in facilitating the degradation of their transcripts (
). Furthermore, the expression of some RNA-binding proteins with preferential binding activity to the U-rich region in RNA has been shown to be implicated in neuronal development inDrosophila, Xenopus, and mouse embryo (
). Whether the protein factor binding to the H-ferritin 3′-UTR would also interact with the U-rich region located in the 3′-UTRs of other genes remains to be clarified.
UV cross-linking experiments revealed that the H-ferritin 3′-UTR-binding protein has a molecular size of ∼43 kDa (data not shown). Recently, a number of pyrimidine tract-binding proteins which participate in RNA splicing or belong to a family of heterogenous nuclear ribonucleoproteins have been cloned and characterized (
). These nuclear proteins have apparent size in the ranges of 56–70 kDa and some of them exhibit preferential binding activity to the sequence rich in U. Based on the differences in subcellular localization, molecular size, and the restriction in cell-type origin, the H-ferritin 3′-UTR-binding protein appears to be distinct from these identified pyrimidine-binding proteins. Nevertheless, a recent study has showed that a protein factor which binds to a U-rich sequence in the 3′-UTR of GAP-43 mRNA shares sequence homology with PTB, an identified pyrimidine tract-binding protein implicated in RNA splicing (
). Whether the 43-kDa protein binding to the H-ferritin 3′-UTR is a PTB-like protein is an intriguing question awaiting to be further investigated. It is apparent that disclosure of the molecular nature of this RNA-binding protein should provide insight into the mechanism underlying the differential regulation of H-ferritin gene expression at the post-transcriptional level in myeloid cells.