Novel translational control through an iron-responsive element by interaction of multifunctional protein YB-1 and IRP2

Abstract

The eukaryotic Y-box-binding protein YB-1 functions in various biological processes, including DNA repair, cell proliferation, and transcriptional and translational controls. To gain further insight into how human YB-1 plays its role in pleiotropic functions, we here used two-hybrid screenings to identify partners of this protein; the results showed that YB-1 itself, iron-regulatory protein 2 (IRP2), and five ribosomal proteins each served as partners to YB-1. We then examined the biological effect of the interaction of YB-1 and IRP2 on translational regulation. Both in vitro binding and coimmunoprecipitation assays showed the direct interaction of YB-1 and IRP2 in the presence of a high concentration of iron. RNA gel shift assays showed that YB-1 reduced the formation of the IRP2-mRNA complex when the iron-responsive element of the ferritin mRNA 5' untranslated region (UTR) was used as a probe. By using an in vitro translation assay using luciferase mRNA ligated to the ferritin mRNA 5'UTR as a reporter construct, we showed that both YB-1 and IRP2 inhibited the translation of the mRNA. However, coadministration of YB-1 and IRP2 proteins abrogated the inhibition of protein synthesis by each protein. An In vivo coimmunoprecipitation assay showed that IRP2 bound to YB-1 in the presence of iron and a proteasome inhibitor. The direct interaction of YB-1 and IRP2 provides the first evidence of the involvement of YB-1 in the translational regulation of an iron-related protein.

FIG. 1.

Interaction of YB-1 with IRP2 in vitro. (A) Schematic illustration of modular domains of IRP2. The positions of cysteines that are thought to be iron sensors are indicated by the letter C and by the numbers under the schema. Abbreviations: IDD domain, iron-dependent degradation domain responsible for the iron-dependent IRP2 degradation; Two-Hybrid clone, plasmids containing partial cDNA of human IRP2 fused to the GAL4 activation domain were rescued from true positive clones from the yeast strain MaV203; Thio-IRP2 and Thio/IRP2ΔN, full or partial IRP2 were cloned into the ThioHis plasmid as described in Materials and Methods. (B to D) GST-pull down assay. (B) 1 μg of GST-YB-1 and GST which were bound to 30 μl of glutathione-Sepharose 4B beads were incubated with a 100-fold excess of Thio-IRP2ΔN. The beads were washed three times with washing buffer containing 1% NP-40. Proteins bound to the beads were resolved by SDS-PAGE and analyzed by Western blotting using an anti-Thio antibody. (C) 1 μg of GST-YB-1 protein which binds to glutathione-Sepharose 4B was incubated with Thio-IRP2ΔN in the absence or presence of 100 U of RNase A. Beads were washed, and then bound proteins were eluted and analyzed as described above. (D) 1 μg of GST-YB-1 protein which binds to glutathione-Sepharose 4B was incubated with full-length Thio-IRP2 in the presence of various concentrations of FAC as indicated. The beads were washed, and then bound proteins were eluted and analyzed as described above.

FIG. 2.

Identification of the IRP2-binding domain in YB-1. (A) Schematic illustration of the full-length and deleted GST-YB-1 proteins used in this study. The striped box indicates the cold shock domain. (B) GST-pull down assay. 1 μg of each GST-YB-1 deletion mutant and GST which binds to 30 μl of glutathione-Sepharose 4B beads were incubated with a 100-fold excess of full-length Thio-IRP2 in the presence or absence of 50 μM FAC. Proteins bound to the beads were resolved by SDS-PAGE and analyzed by Western blotting using an anti-Thio antibody.

FIG. 3.

Identification of the YB-1-binding domain in IRP2. (A) Schematic illustration of the ThioHis-IRP2 deletion mutants used in this study. (B) Expression of recombinant proteins. Deletion mutants were expressed in bacteria and analyzed by Coomassie brilliant blue staining of SDS-PAGE gels. (C) GST pull-down assay. 1 μg of GST-YB-1 or GST which binds to 30 μl of glutathione-Sepharose 4B beads was incubated with a 100-fold excess of ThioHis-IRP2 deletion mutants. Proteins bound to the beads were resolved by SDS-PAGE and analyzed by Western blotting using an anti-Thio antibody.

FIG. 4.

YB-1 inhibits IRP2 binding to RNA. (A) The indicated amounts of GST and GST fusion IRP2 were incubated with 5 × 10⁴ cpm of ³²P-labeled ferritin IRE for 15 min. IRP2-specific antibody (Ab) was added to lanes 5 and 10. IRP2-IRE complexes were separated by REMSA using a 6% native polyacrylamide gel. (B) 5 pmol of GST-IRP2 was incubated with the indicated concentration of GST or GST-YB-1 for 10 min and with 5 × 10⁴ counts of ³²P-labeled ferritin IRE for another 15 min. Samples were assessed by REMSA.

FIG. 5.

IRP2 or YB-1 represses translation. (A) Schematic illustration of the reporter plasmid containing luciferase cDNA ligated to the ferritin heavy-chain mRNA 5′ UTR region 300 bp (ferritin 5′UTR-luciferase) or containing unligated luciferase cDNA (luciferase). (B and C) Each reporter plasmid was transcribed in vitro, which was driven by T7 RNA polymerase and then translated in the rabbit reticulocyte lysate system. The indicated amount of GST-IRP2 was added to the system with 2 μg of ferritin 5′ UTR-luciferase RNA (B) or luciferase RNA (C). The luciferase assay was performed after incubation for 1.5 h at 30°C. All data are shown as the mean ± standard deviation from three independent experiments. (D and E) The indicated amount of GST-YB-1 was added to the reticulocyte lysate system with 2 μg of ferritin 5′UTR-luciferase RNA (D) or luciferase RNA (E).

FIG. 6.

Functional interactions between YB-1 and IRP2 were revealed by their effects on the ferritin 5′ UTR-containing mRNA. (A) The indicated amounts of GST-IRP2 and GST-YB-1 were added to the rabbit reticulocyte lysate system using 2 μg of ferritin 5′ UTR-luciferase RNA. The in vitro translation was performed for 1.5 h at 30°C, and the luciferase assays were performed after incubation. Data are shown as the mean and standard deviation of three independent experiments. ∗, Analyzed by Student's t test, P < 0.01. (B) Coadministration of GST-YB-1 and GST-IRP2 proteins in the presence of DFO.

FIG. 7.

Flag-IRP2 binds YB-1 in a rabbit reticulocyte lysate. Rabbit reticulocyte lysate incubated with pCMV/Flag-IRP2 plasmid or control vector was immunoprecipitated (IP) with either control IgG (lane 5) or anti-Flag M2-agarose (lanes 2 to 4) in the presence of DFO (lane 3) or FAC (lane 4). The immunocomplexes were separated by SDS-PAGE and transferred to an Immobilon-P membrane. Blots were probed with anti-YB-1 antibody (Ab).

FIG. 8.

Coimmunoprecipitation of IRP2 and YB-1 from cell extracts. RAW264.7 cells were incubated in 100 μM DFO (lane 1), 600 μM FAC (lane 2), or FAC and MG132 (1 μM) (lane 3) for 6 h. Extracts from each treated cell were immunoprecipitated (IP) with immune anti-IRP2 (lanes 1-3) or control mouse IgG (lane 4). The immunocomplex was subjected to SDS-PAGE and transferred to an Immobilon-P membrane. Blots were probed with anti-YB-1 antibody (Ab). YB-1 is indicated by an asterisk.