Delayed Translational Silencing of Ceruloplasmin Transcript in Gamma Interferon-Activated U937 Monocytic Cells: Role of the 3′ Untranslated Region

Reagents.

Rabbit reticulocyte lysate, methionine-minus amino acid mixture, and RNasin were purchased from Promega (Madison, Wis.). Puromycin, cycloheximide, and other assay reagents were obtained from Sigma (St. Louis, Mo.). Human IFN-γ was from Life Technologies (Gaithersburg, Md.). [³⁵S]methionine was purchased from NEN-DuPont (Boston, Mass.) for in vitro translation (translation grade) and from ICN (Costa Mesa, Calif.) for metabolic labeling (Trans-label). Purified human Cp was obtained from Calbiochem (La Jolla, Calif.).

Cultured cells.

U937 cells (American Type Culture Collection, Rockville, MD; CRL 1593.2) were preincubated for 3 h in serum-free RPMI 1640 medium (10⁸ cells per 50 ml) before addition of IFN-γ (500 U/ml). HT1080, HeLa, and HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium containing 5% fetal bovine serum, and human umbilical vein endothelial cells were cultured in MCDB 107 medium containing 15% fetal bovine serum. After overnight incubation of the cells, the medium was replaced with serum-free medium and incubated for 3 h before IFN-γ treatment.

Immunoblot analysis.

Conditioned medium from 8 × 10⁶ U937 cells was centrifuged at 14,000 × g, and the supernatant was concentrated by ultrafiltration with Centricon-30 filters (Amicon, Beverly, Mass.). The concentrate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7% polyacrylamide) (SDS-PAGE) with Protogel (National Diagnostics, Atlanta, Ga.) and transferred by a semidry method to an Immobilon-P membrane (Millipore, Bedford, Mass.). The membrane was incubated with polyclonal rabbit anti-human Cp immunoglobulin G (IgG) (1:20,000; Accurate Chemical, Westbury, N.Y.) as a primary antibody and then with peroxidase-conjugated secondary antibody (1:10,000; Boehringer Mannheim, Indianapolis, Ind.). The blot was developed by chemiluminescence by using the ECL (Amersham, Arlington Heights, Ill.) enhanced chemiluminescence system and XAR-5 film (Kodak, Rochester, N.Y.). Immunoblots were quantitated densitometrically with a Microtek III flatbed scanner and the N.I.H. Image program, provided by Wayne Rasband, National Institutes of Mental Health.

Metabolic labeling.

U937 cells (8 × 10⁶ cells in 4 ml of RPMI 1640 medium) were treated with IFN-γ (500 U/ml) for up to 24 h. The cells were collected by centrifugation at 7,000 × g, resuspended, and metabolically labeled by incubation for 2 h with [³⁵S]methionine (100 μCi/ml; Trans-Label) in methionine-free medium. The cells were pelleted by centrifugation at 7,000 × g, and the conditioned medium was collected. To prepare lysates, the cells were suspended in a mixture of 50 mM Tris (pH 7.6), 50 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), and 0.5% NP40; subjected to three freeze-thaw cycles; and passed several times through a 26-gauge needle. Newly synthesized, ³⁵S-labeled Cp was immunoprecipitated from conditioned medium and lysates by using rabbit anti-human Cp IgG and protein A-Sepharose in buffer containing 50 mM Tris (pH 7.6), 150 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM PMSF. Proteins were resolved by SDS-PAGE (7% polyacrylamide), and the gel was fixed and treated with Amplify (Amersham, Arlington Heights, Ill.) and then dried and allowed to expose MR film (Kodak, Rochester, N.Y.).

RNA blot analysis.

Treated U937 cells (10⁸ cells) were collected by centrifugation, and total RNA was extracted with Trizol reagent (Life Technologies) according to the manufacturer’s instructions and subjected to poly(A) selection with an OligoTex mRNA kit (Qiagen, Stanford, Calif.). The mRNA isolated from 100 μg of total RNA was fractionated on a 1% agarose–formaldehyde gel and transferred to Nytran membranes (Schleicher and Schuell, Keene, N.H.). The blot was hybridized with a random primer-labeled 646-bp human Cp cDNA probe (nucleotides [nt] 984 to 1629 in the open reading frame). The blot was stripped and rehybridized with full-length glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or human γ-actin cDNA probes.

Cloning of the 3′-UTR of human Cp.

For studies of the human Cp 5′-flanking region not described here, a human genomic library in the bacterial artificial chromosome vector pBeloBAC11 (Research Genetics, Huntsville, Ala.) was examined by PCR screening with primers corresponding to the extremes of Cp exon 1 (bp 1 to 25 and 117 to 140) (13). A pool of putative positive clones was rescreened by PCR with anchor primers in exon 1, and a single positive clone was isolated. DNA sequencing with an exon 1-specific primer 40 bp downstream of the start codon gave a sequence identical to the known Cp exon 1 sequence, demonstrating its authenticity. The same clone was sequenced with a Cp exon 19-specific primer (13) and gave a sequence which was identical to the published 247-bp Cp 3′-UTR (56), except for substitution of an A for a G in position 13 and TTG in place of GCC in positions 164 to 166; identical results were obtained from multiple sequencing reactions of three individual clones. The entire 247-bp Cp 3′-UTR was subcloned into pcDNA3 downstream of the T7 promoter (pcDNA3/Cp 3′-UTR).

Synthesis of deletion fragments of Cp 3′-UTR.

The 247-bp 3′-UTR was excised from pcDNA3/Cp 3′-UTR by digestion with BamHI and XhoI. The insert was purified by gel electrophoresis and amplified by PCR with Pfu polymerase and primers appropriate for desired segment; the 5′ primer also contained the T7 promoter sequence upstream of the Cp-specific sequence. The PCR products were gel purified, sequenced, and used as a template to generate the corresponding RNA segments by in vitro transcription with T7 RNA polymerase by using Megascript (Ambion, Austin, Tex.). Finally, the transcripts were purified on a 5% acrylamide–8 M urea gel. The synthetic Cp 3′-UTR segments are designated 1–247, 51–247, 101–247, 1–200, 1–150, and 1–100, where 1 is the first nucleotide after the stop codon and 247 is the last nucleotide before the poly(A) tail.

Isolation of polysomal mRNA.

Polysomal mRNA was isolated from U937 cells as described previously (54). In brief, U937 cells (5 × 10⁸ cells) were treated with IFN-γ for up to 24 h and homogenized in 5 ml of polysome buffer consisting of 20 mM Tris (pH 7.4), 10 mM MgCl₂, 300 mM KCl, 10 mM DTT, 100 U of RNasin per ml, and 100 μg of cycloheximide per ml, followed by centrifugation at 10,000 × g for 15 min. The postmitochondrial supernatant was layered over a sucrose (20% [wt/vol]) cushion containing cycloheximide and centrifuged at 149,000 × g for 2 h. The polysome-containing pellet was collected, and the nonpolysomal fraction was obtained by ethanol precipitation of the supernatant. Both fractions were subjected to SDS-proteinase K digestion.

Preparation of cell extracts.

U937 cells (10⁸ cells), or in some experiments, HeLa, HT1080, HepG2, and human umbilical vein endothelial cells, were treated with IFN-γ, harvested by scraping, and suspended in a mixture of 50 mM Tris (pH 7.6), 50 mM NaCl, 1 mM PMSF, and 1 mM DTT. The suspension was subjected to three freeze-thaw cycles, was passed several times through a 26-gauge needle, and was ultracentrifuged at 100,000 × g for 30 min. The protein concentration of the supernatant was adjusted to 1 mg/ml, and 4 μg was used in the in vitro translation reaction.

In vitro translation of Cp mRNA by reticulocyte lysate.

Total RNA from U937 cells (10⁸ cells) was isolated by two rounds of Trizol extraction. An aliquot (100 μg) was subjected to in vitro translation by addition of rabbit reticulocyte lysate, 20 μM a methionine-free amino acid mixture, 40 U of RNasin, and 20 μCi of translation-grade [³⁵S]methionine in 50 μl for 1 h at 30°C. A 45-μl aliquot was subjected to immunoprecipitation with rabbit anti-human Cp IgG and protein-A Sepharose in buffer containing 50 mM Tris, 150 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM PMSF (pH 7.6). Immunoprecipitated protein was resolved by SDS-PAGE (7% polyacrylamide). The gel was fixed, soaked in Amplify, dried, and allowed to expose Kodak MR film. To evaluate the total pool of newly synthesized proteins, a 5-μl aliquot that was not subjected to immunoprecipitation was similarly resolved by SDS-PAGE (7% polyacrylamide) and fluorography. In decoy experiments, 500 ng of synthetic unlabeled transcript of Cp 3′-UTR, 15-lipoxygenase 3′-UTR, Cp exon 5, and deletion fragments of the Cp 3′-UTR were preincubated for 10 min with the cell extract before being added to the in vitro translation reaction.

In vitro transcription of Cp 3′-UTR.

A synthetic transcript of the 247-nt Cp 3′-UTR was prepared by in vitro transcription of XbaI-linearized pcDNA3/Cp 3′-UTR using T7 polymerase in the presence of [α-³²P]UTP (MaxiScript kit; Ambion). The full-length transcript was purified by electrophoresis on a 5% acrylamide gel containing 8 M urea. Unlabeled synthetic transcripts of the Cp 3′-UTR, the 241-nt 15-lipoxygenase 3′-UTR, and the 255-nt Cp exon 5 were prepared by in vitro transcription of pcDNA3/Cp 3′-UTR, pBS[SK(10R)] (42, 43), and pcDNA3/Cp exon 5, respectively, and were gel purified.

RNA gel shift assay.

[α-³²P]UTP-labeled Cp 3′-UTR (10 fmol) was incubated for 30 min at room temperature with U937 cell extract (10 μg of protein) in 20 μl of reaction buffer containing 12 mM HEPES (pH 8.0), 15 mM KCl, 0.25 mM DTT, 5 mM MgCl₂, 0.1 mM PMSF, 200 μg of yeast tRNA per ml, 40 U of RNasin, and 10% glycerol. In competition experiments, a 25-fold molar excess of unlabeled Cp 3′-UTR and 25- or 100-fold molar excess of Cp 3′-UTR segments were added to the extract 10 min before the addition of radiolabeled probe. RNA-protein complexes were resolved by native gel electrophoresis (5% polyacrylamide in 0.5× Tris-buffered EDTA). The gel was dried, and the retarded probe was visualized by autoradiography.

Termination of Cp synthesis in the presence of abundant Cp mRNA.

The temporal relationship between Cp synthesis and Cp mRNA levels in IFN-γ-treated U937 cells was examined. The steady-state level of Cp mRNA was measured by Northern blot analysis and was normalized by comparison to GAPDH mRNA (Fig. 1A and B). In agreement with previous results (35), maximum Cp mRNA was reached after about 8 h and remained high for at least 24 h (Fig. 1D). Cp synthesis and secretion during 4- or 8-h intervals were measured by Western blot analysis (Fig. 1C). During the first 8 h, Cp synthesis was nearly proportional to Cp mRNA (Fig. 1D). At later times, Cp synthesis decreased disproportionately compared to Cp mRNA; between 16 and 24 h, there was essentially no Cp synthesis despite the presence of abundant Cp mRNA. To confirm the low rate of synthesis at late times after IFN-γ treatment, de novo Cp synthesis was measured by pulse-labeling of cells with [³⁵S]methionine. Labeled Cp in the conditioned medium and lysate was determined by immunoprecipitation with rabbit anti-human Cp IgG, SDS-PAGE, and autoradiography. The lysate and conditioned medium of cells treated with IFN-γ for 8 h contained abundant newly synthesized Cp, but essentially no new Cp was detected in either pool after treatment of cells for 24 h (Fig. 1E).

Mechanism of translational silencing of Cp.

The low translation rate of Cp mRNA was consistent with either a modified and untranslatable transcript or translational silencing of an intact transcript. To test transcript integrity, U937 cells were treated with IFN-γ for up to 24 h, and total RNA was isolated and subjected to cell-free translation by a rabbit reticulocyte lysate in the presence of [³⁵S]methionine. Newly translated, radiolabeled Cp was detected by immunoprecipitation with anti-Cp IgG and fluorography. The in vitro translational efficiencies of Cp mRNA derived from cells treated with IFN-γ for from 4 to 24 h were nearly identical (Fig. 2). The translated Cp product comigrated with an authentic human Cp standard, as shown by Coomassie blue staining (not shown). In control experiments, the Cp band was not seen with rabbit IgG in the immunoprecipitation reaction, and background translation in the absence of added RNA was negligible (not shown). The amount of RNA used gave a rate of Cp translation within the linear range of the reticulocyte lysate system (not shown). These results suggest that the low Cp translation rate in U937 cells treated with IFN-γ for 24 h is not due to a defective Cp transcript, but rather is due to a cellular defect in Cp translation.

Transcript-specific translational control often involves translocation of regulated transcripts between two defined pools: an inactive, nonpolysomal pool containing cytoplasmic messenger ribonucleoprotein particles and an active polyribosomal pool of mRNA-ribosome complexes. We thus investigated whether inefficient Cp translation was due to a defect in the association of Cp mRNA with polyribosomes. U937 cells were treated with IFN-γ for 8 or 24 h, cell homogenates were separated into polysomal and nonpolysomal fractions by centrifugation through a sucrose cushion, and Cp mRNA was detected by Northern analysis. After IFN-γ treatment for 8 h (when the rate of Cp protein synthesis was high) Cp mRNA was primarily associated with the polysomal fraction (Fig. 3A). In contrast, after IFN-γ treatment for 24 h (when Cp synthesis was low), Cp mRNA was found almost exclusively in the inactive nonpolysomal fraction. In a control experiment, puromycin was added to the 8-h fractions to release ribosomes from mRNA (54); puromycin shifted the Cp transcript into the nonpolysomal fraction, showing that the presence of Cp in the polysomal fraction was not due to nonspecific interactions or aggregate formation (Fig. 3A). Reprobing the same blot with human γ-actin cDNA showed that this transcript was not shifted from the polysomal to nonpolysomal fraction during prolonged treatment with IFN-γ, indicating at least partial transcript specificity of the translocation process (Fig. 3B).

Translational silencing of Cp transcript by a cellular factor.

Transcript-specific translational control in eukaryotes generally involves binding of inhibitory trans-acting factors to specific mRNA sequences. To investigate the presence of inhibitory factors, extracts were made from IFN-γ-treated U937 cells and tested for their ability to block Cp mRNA translation in cell-free reticulocyte lysates. Extracts made from cells treated with IFN-γ for 24 h completely inhibited in vitro translation of Cp mRNA derived from cells treated with IFN-γ for 8 h (Fig. 4A). In contrast, extracts made from untreated cells or from cells treated with IFN-γ for 8 h were not inhibitory, showing specificity with respect to the extract source. Similar results were obtained with RNA isolated from cells treated with IFN-γ for 24 h, verifying that this transcript is regulatable as well as translatable (Fig. 4A). The transcript specificity of the inhibitor in the 24-h extract was investigated by analysis of the translated products before immunoprecipitation with anti-Cp IgG. The lack of inhibition of translation of other major U937 cellular proteins indicates a high degree of transcript specificity (Fig. 4B). In a control experiment, we tested the possibility that the 24-h extracts did not inhibit translation of Cp mRNA, but instead increased its rate of degradation. Total RNA from cells treated with IFN-γ for 8 h was incubated with rabbit reticulocyte lysate in the presence of extracts from cells treated with IFN-γ for 8 and 24 h. The RNA was reisolated and subjected to poly(A) selection and Northern blot hybridization with a Cp cDNA probe. Neither extract caused measurable Cp mRNA degradation (Fig. 4C), consistent with a factor in the 24-h extract that represses translation.

To investigate cell-type specificity, the presence of translational inhibitory activity in extracts of several cell lines was examined. HT1080, HeLa, HepG2, and human umbilical vein endothelial cells were treated with IFN-γ for 24 h, and extracts were prepared. All of these cells contain IFN-γ receptors, and HT1080 and HeLa cell lines are commonly used in studies of IFN-γ signaling. HepG2 cells were chosen since IFN-γ stimulates (albeit modestly) Cp synthesis by these cells (35). None of these extracts inhibited the cell-free translation of Cp mRNA isolated from U937 cells after 8 h of IFN-γ treatment (Fig. 5A). Since U937 cells are a human promonocytic cell line, we examined the activity of extracts from freshly isolated human peripheral blood monocytes. These extracts inhibited Cp translation, suggesting a specificity for cells of myeloid origin (Fig. 5B).

Role of Cp 3′-UTR in translational Control by IFN-γ.

Assuming that the human Cp 5′-UTR has a length comparable to the 30-nt 5′-UTR of rat Cp (19), this transcript region is unlikely to provide secondary structure necessary for translational control. To test the role of the Cp 3′-UTR in translational inhibition, we tested whether a synthetic Cp 3′-UTR, added in excess as a decoy, could overcome the translational silencing activity of putative RNA-binding proteins. The synthetic Cp 3′-UTR almost completely restored translation in the presence of inhibitory extracts from U937 cells treated with IFN-γ for 24 h (Fig. 6). As a control to show specificity of the Cp 3′-UTR, a synthetic transcript of Cp exon 5 was found to have no effect on the translational inhibition by the cell extract. In the same experiment, we showed that the 15-lipoxygenase 3′-UTR, known to contain a regulatory region responsible for translational silencing of that transcript (42, 43), also was ineffective (Fig. 6). To determine the region of the Cp 3′-UTR responsible for translational control, we measured the ability of deletion fragments of the UTR (Fig. 7, top [schematic]) to overcome the silencing activity of the 24-h extract. Cp 3′-UTR segments 51–247, 1–200, and 1–150 were as effective as the full-length UTR, but fragment 1–100 had only marginal activity, and 101–247 was completely inactive (Fig. 7, bottom). These results suggest that nt 50 to 150 are critical for Cp translational control by IFN-γ.

Translational control generally results from an interaction between cellular RNA-binding proteins and specific cis-acting sites in transcript UTRs. We therefore investigated the presence of a factor(s) in extracts of IFN-γ-treated U937 cells that bind to the Cp 3′-UTR. Interacting proteins were determined by an RNA electrophoretic mobility shift assay by using [α-³²P]UTP-labeled Cp 3′-UTR as probe. The probe was retarded by extracts from U937 cells treated with IFN-γ for 24 h (Fig. 8). Probe-binding complexes were not detected in extracts from untreated cells or from cells treated with IFN-γ for 8 h. The specificity of the RNA-protein interaction was shown by efficient competition by an excess of unlabeled Cp 3′-UTR and also by the lack of competition by the synthetic 15-lipoxygenase 3′-UTR or by RNA corresponding to Cp exon 5.

To investigate whether the complex observed in the RNA gel shift assay may be responsible for the translational silencing activity in the 24-h extract, the complex-forming activity of the Cp 3′-UTR was mapped and compared to the map of the silencing activity. Formation of the complex with the deletion fragments of the Cp 3′-UTR was measured as competition by unlabeled fragments for binding of the complex to radiolabeled full-length 3′-UTR. UTR segments 51–247, 1–200, and 1–150 were as effective competitors as unlabeled, full-length UTR, but segments 1–100 and 101–247 did not compete for binding even at a 25-fold molar excess (Fig. 9). The segments gave essentially the same results in the two assay systems; namely, only those segments that competed for binding to complexes when incubated with extracts also overcame the translational silencing activity when added as decoys (Fig. 7, top). The minor difference between the relative activities of segments 1–100 and 101–247 in the two assays may be due to the dissimilar experimental conditions or the nonquantitative nature of the assays. Overall, these results are consistent with the presence of a single factor (or complex) that binds to the Cp 3′-UTR and translationally silences the transcript.