Article Figures & Data
Figures
- Fig. 1.
Models for translational silencing of Cp. (A) “Circular” or “closed-loop” mRNA model showing circularization mediated by PABP binding to both the poly(A) tail and eIF4G of the initiation complex. (B) Inhibition of Cp mRNA translation by disruption of Cp mRNA circularization. Indicated are two potential mechanisms by which the translational inhibitor of Cp (CpTI) may block translation: 1, disruption of PABP interaction with poly(A); 2, disruption of PABP interaction with eIF4G. (C) Inhibition of Cp mRNA translation by a mechanism dependent on mRNA circularization. In this proposed mechanism (mechanism 3), circularization of the transcript brings the 3′-UTR-bound CpTI into the proximity of the 5′-translation initiation complex, where it exerts its inhibitory activity. Abbreviations: eIF4A, eukaryotic translation initiation factor 4A; eIF4E, eukaryotic translation initiation factor 4E; eIF3, eukaryotic translation initiation factor 3; ORF, open reading frame.
- Fig. 2.
Role of 3′-UTR and the poly(A) tail in translational silencing of Cp in IFN-γ-activated U937 cells. (A) U937 cells (5 × 108) were treated with IFN-γ (500 U/ml) for 8 or 24 h. For the translation template, total RNA was extracted from cells treated with IFN-γ for 8 h. The mRNA from 100 μg of total RNA was subjected to in vitro translation in a reticulocyte lysate with [35S]methionine in the presence of cytosolic extracts (4 μg of protein) from U937 cells treated with IFN-γ for 8 or 24 h. Some extracts were preincubated with synthetic, unlabeled Cp 3′-UTR and 15-lipoxygenase (15-LO) 3′-UTR cRNA (0.5 μg) as competitors before being added to the translation reaction mixture. An aliquot (45 μl) was subjected to immunoprecipitation (IP) using rabbit anti-human Cp IgG and translated, and [35S]Cp was resolved by SDS-PAGE (7% polyacrylamide) and detected by fluorography. (B) Total in vitro protein synthesis was determined with a 5-μl aliquot of the same translation reaction mixture described in panel A that was not subjected to immunoprecipitation. 35S-labeled protein was resolved by SDS-PAGE and detected by fluorography. (C) Capped cRNA transcripts cap-Luc, cap-Luc-Cp 3′-UTR, and cap-Luc-Cp 3′-UTR–poly(A) were gel purified and subjected (200 ng of each) to in vitro translation at 30°C for 60 min in a rabbit reticulocyte lysate containing [35S]methionine and cytosolic extracts (4 μg of protein) from U937 cells treated with IFN-γ for 8 or 24 h. A capped, gel-purified transcript of T7 gene 10 (100 ng) was added to each lysate as a loading control. Newly translated,35S-labeled Luc and T7 gene 10 were resolved by SDS-PAGE (7% polyacrylamide) and detected by fluorography. (D) The relative rate of Luc synthesis under each condition was quantitated by densitometry.
- Fig. 3.
Role of poly(A) tail in translational silencing of endogenous Cp mRNA. U937 cells (5 × 108) were treated with IFN-γ (500 U/ml) for 8 or 24 h. Poly(A)-containing mRNA was isolated from total RNA (100 μg) extracted from cells treated for 8 h. The poly(A) tail was removed by incubation with oligo(dT) (18-mer), and then double-stranded regions of DNA-RNA hybrids were digested by incubation with RNase H. The reaction was terminated by addition of 10 mM EDTA followed by ethanol precipitation. (A) The Cp transcript length was determined by Northern blot hybridization using radiolabeled Cp cDNA as probe. The two major transcripts are indicated by arrow. (B) To verify the absence of a poly(A) tail, aliquots of RNase H-treated and untreated cellular mRNA were subjected to reverse transcription using Superscript and oligo(dT) followed by PCR amplification using primers encompassing the full-length Cp 3′-UTR. (C) Intact and deadenylated cellular mRNA were subjected to in vitro translation in a rabbit reticulocyte lysate with [35S]methionine in the presence of cytosolic extracts (4 μg of protein) from U937 cells treated with IFN-γ for 8 or 24 h (the rightmost pair of lanes show the effect of replicate 24-h extracts on translation of deadenylated RNA). Newly synthesized, [35S]Cp was immunoprecipitated (IP) with rabbit anti-human Cp IgG, resolved by SDS-PAGE, and detected by fluorography (arrow). (D) To show specificity of the translational inhibition by U937 cell extracts, aliquots of the rabbit reticulocyte lysates that were not subjected to immunoprecipitation were resolved by SDS-PAGE and fluorography.
- Fig. 4.
Effect of the cytosolic inhibitor on binding of PABP to polyadenylated chimeric transcript. (A) The recognition site of anti-human PABP antibody was mapped by expression of Flag-tagged chimeric plasmids containing N- and C-terminal PABP regions in Hela cells. Cells were infected with vaccinia virus vTF7-3 and transiently transfected with pcDNA3-Flag-PABP(1–376), pcDNA3-FLAG-PABP(377–633), or with pcDNA3-Flag as control. At 20 h after transfection, cell extracts (10 mg protein) were resolved by SDS-PAGE (10% polyacrylamide) and subjected to immunoblot analysis with anti-human PABP antibody (left) or anti-Flag antibody (right). (B) (Upper panel) Gel-purified transcripts cap-Luc-Cp 3′-UTR and cap-Luc-Cp 3′-UTR–poly(A) (200 ng of each) were incubated with rabbit reticulocyte lysates for 15 min in the presence of cytosolic extracts from U937 cells treated with IFN-γ for 8 or 24 h. PABP was immunoprecipitated (IP) by addition of monoclonal anti-human PABP (3 μl) and protein A-Sepharose. PABP-bound RNA was extracted by Trizol and subjected to reverse transcription using a primer for the extreme 3′ end of Cp 3′-UTR followed by PCR amplification with primers for the extreme 5′ and 3′ ends of the full-length Cp 3′-UTR (the 17 cycles used gave a product in the linear range of the assay [not shown]). The leftmost lane is a DNA ladder containing a series of DNA fragments at multiples of 100-bp (Life Technologies). The predicted position of the amplified product is indicated by an arrow. (Lower panel) Same as in the upper panel but without the addition of reverse transcriptase (RT) to the reverse transcription reaction. (C) (Upper panel) The binding of PABP to endogenous Cp transcript in U937 cells was determined. U937 cells were incubated with IFN-γ for 8 or 24 h, and lysates (400 μg of protein) were subjected to immunoprecipitation using anti-human PABP antibody. PABP-bound mRNA was extracted and subjected to reverse transcription using an oligo(dT) primer. The cDNA was subjected to 12 or 16 cycles of PCR amplification using primers for the extreme 5′ and 3′ ends of the full-length Cp 3′-UTR. (Lower panel) Same as in the upper panel but without the addition of reverse transcriptase.
- Fig. 5.
Effect of the cytosolic inhibitor on binding of PABP to eIF4G. Reticulocyte lysates (50 μl) were incubated with cytosolic extracts (4 μg of protein) from U937 cells treated with IFN-γ for 8 or 24 h. To one set of lysates was added cap-Luc-Cp 3′-UTR–poly(A) cRNA (200 ng). PABP was immunoprecipitated (IP) with monoclonal anti-human PABP antibody (Ab) (or with monoclonal antibody [mAb] Sp2/O as a control) and protein A-Sepharose beads. The beads were washed and boiled with Laemmli buffer, and the samples were subjected to SDS-PAGE (5% polyacrylamide). The resolved proteins were transferred to Immobilon-P and subjected to immunoblot analysis using polyclonal rabbit anti-human eIF4G antibody. The rightmost lane contained untreated reticulocyte lysate (1 μl) as an eIF4G standard.
- Fig. 6.
Requirement for PABP in transcript-specific translational silencing of the polyadenylated chimeric reporter transcript. (Upper panel) Rabbit reticulocyte lysates were preincubated for 15 min with 3 μl of monoclonal anti-human PABP or with monoclonal antibody (mAb) Sp2/O as a control. Gel-purified cRNA transcript cap-Luc-Cp 3′-UTR–poly(A) (100 ng) was subjected to in vitro translation by reticulocyte lysates for 60 min at 30°C in the presence of [35S]methionine and cytosolic extracts (4 μg of protein) from IFN-γ-treated U937 cells. A cRNA transcript encoding T7 gene 10 (100 ng) was added as a control. Newly synthesized,35S-labeled Luc and T7 gene 10 were resolved by SDS-PAGE, and the radiolabeled bands were detected by fluorography. (Lower panel) The relative amount of Luc synthesis was quantitated by densitometry and normalized by division by T7 gene 10 synthesis under each condition.
- Fig. 7.
Requirement for eIF4G in the transcript-specific translational silencing of the polyadenylated chimeric reporter transcript. (Upper panel) A capped transcript containing luciferase upstream of the Cp 3′-UTR and a 30-nt poly(A) tail [cap-Luc-Cp 3′-UTR–poly(A)] was prepared by in vitro transcription in the presence of the cap analog, m7G(5′)ppp(5′)G. Rabbit reticulocyte lysates were preincubated for 15 min with 1 μl of polyclonal rabbit antiserum against human eIF4G or rabbit anti-GST antiserum as a control. Gel-purified cRNA transcript (100 ng) was subjected to in vitro translation in the presence of [35S]methionine and cytosolic extracts (4 mg of protein) from IFN-γ-treated U937 cells for 60 min at 30°C. A cRNA transcript encoding T7 gene 10 (100 ng) was simultaneously translated as a control. Newly synthesized, 35S-labeled luciferase and T7 gene 10 were resolved by SDS-PAGE, and the radiolabeled bands were detected by fluorography (indicated by arrows). (Lower panel) The relative rate of Luc synthesis was quantitated by densitometry and normalized by division by T7 gene 10 synthesis under each condition. Ab, antibody.
