Subcellular relocalization of a trans-acting factor regulates XIAP IRES-dependent translation

Abstract

Translation of the X-linked inhibitor of apoptosis (XIAP) proceeds by internal ribosome entry site (IRES)-mediated initiation, a process that is physiologically important because XIAP expression is essential for cell survival under conditions of compromised cap-dependent translation, such as cellular stress. The regulation of internal initiation requires the interaction of IRES trans-acting factors (ITAFs) with the IRES element. We used RNA-affinity chromatography to identify XIAP ITAFs and isolated the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). We find that hnRNP A1 interacts with XIAP IRES RNA both in vitro and in vivo and that hnRNP A1 negatively regulates XIAP IRES activity. Moreover, XIAP IRES-dependent translation is significantly reduced when hnRNP A1 accumulates in the cytoplasm. Osmotic shock, a cellular stress that causes cytoplasmic accumulation of hnRNP A1, also leads to a decrease in XIAP levels that is abrogated by knockdown of hnRNP A1 expression. These results suggest that the subcellular localization of hnRNP A1 is an important determinant of its ability to negatively regulate XIAP IRES activity, suggesting that the subcellular distribution of ITAFs plays a critical role in regulating IRES-dependent translation. Our findings demonstrate that cytoplasmic hnRNP A1 is a negative regulator of XIAP IRES-dependent translation, indicating a novel function for the cytoplasmic form of this protein.

Figure 1.

hnRNP A1 interacts with the XIAP IRES. (A) RNA-affinity chromatography isolation of XIAP IRES-binding proteins. Precleared protein extracts from HEK293T cells were incubated with either agarose beads coated with XIAP IRES RNA, agarose beads coated with HIAP2 IRES RNA, or agarose beads alone. After protein binding, beads were washed extensively and pelleted, and proteins were eluted by boiling and resolved by SDS-PAGE. Proteins were visualized with Sypro Ruby stain, and the indicated protein species were excised from the gel and subjected to mass spectrometry analysis. (B) The p37 protein is hnRNP A1. Underlined peptide sequences were identified by mass spectrometry analysis of the p37 protein species isolated by RNA-affinity chromatography using XIAP IRES RNA as an affinity matrix. (C) hnRNP A1 associates with XIAP IRES RNA in vitro. XIAP IRES RNA and HIAP2 IRES RNA were used in RNA-affinity chromatography as described in A; isolated proteins were separated by SDS-PAGE, transferred to PVDF membrane, and probed with anti-hnRNP A1 antibody. (D) hnRNP A1 associates with XIAP mRNA in vivo. RNA–protein complexes were cross-linked with formaldehyde, isolated from cells, and immunoprecipitated using antibodies against hnRNP A1, La, and GAPDH. After immunoprecipitation, RNA–protein cross-links were reversed, and the RNA was isolated and used in an RT-PCR reaction with XIAP- and actin-specific oligonucleotide primers.

Figure 2.

hnRNP A1 binds directly to XIAP IRES RNA within the core RNP-binding site. (A) GST (lane 1) or increasing amounts of GST-hnRNP A1 (lanes 2–4) were incubated with ³²P-labeled XIAP IRES RNA probe, UV-cross-linked, and then separated by SDS-PAGE and visualized by autoradiography. (B) GST-hnRNPA1 was incubated with ³²P-labeled XIAP IRES RNA probe alone (No competitor) or a combination of ³²P-labeled XIAP IRES RNA probe and increasing amounts of excess unlabeled (“cold”) competitor RNA molecules (XIAP IRES RNA and HIAP2 IRES RNA), UV-cross-linked, separated by SDS-PAGE, and visualized by autoradiography. (C) hnRNP A1 binding curve for XIAP IRES and HIAP2 IRES RNA. Nitrocellulose filter binding assays were performed and analyzed as described in Materials and Methods. Filter-bound RNA (■, XIAP IRES; ▴, HIAP2 IRES) is plotted as a function of protein concentration. The data presented represent the mean ± SD of three independent experiments. (D) GST-hnRNPA1 was incubated with ³²P-labeled XIAP IRES RNA probe alone (No competitor) or a combination of ³²P-labeled XIAP IRES RNA probe and 100-fold excess of unlabeled competitor RNA molecules, UV-cross-linked, separated by SDS-PAGE, and visualized by autoradiography. Gray boxes in the sequence schematic indicate hnRNP A1–binding sites (labeled as site A and site B).

Figure 3.

Cytoplasmic localization of hnRNP A1 reduces XIAP IRES activity. (A) Immunohistochemistry analysis of HEK293T cells transfected with FLAG-hnRNP A1 or the FLAG-hnRNP A1 F1 mutant using anti-FLAG antibodies; nuclei were visualized by Hoechst staining. (B) hnRNP A1 reduces XIAP IRES activity. HEK293T cells were cotransfected with a plasmid expressing GFP, FLAG-hnRNP A1, or FLAG-hnRNP A1 F1 mutant and the pβgal/5′(−162)/CAT bicistronic reporter plasmid. 48 h after transfection βgal and CAT protein expression was assayed; relative IRES activity is expressed as a ratio of CAT/βgal. The activity of XIAP IRES in GFP transfected cells was set as 100. Mean ± SEM (bars) of three independent experiments performed in triplicate. Expression levels of FLAG-hnRNP A1 and the FLAG-hnRNP A1 F1 mutant were determined by Western-blot analysis using anti-hnRNP A1 antibodies. The asterisk (*) indicates the FLAG-tagged protein species. (C) Overexpression of hnRNP A1 does not affect the integrity of the bicistronic RNA transcript produced from pβgal/5′(−162)/CAT. Cells were treated as described in B; total RNA was isolated 48 h after transfection, and cDNA was generated by reverse transcription. Quantitative PCR was used to determine the levels of βgal and CAT cistrons; values are expressed as CAT relative to βgal (2^{−[Ct(CAT) − Ct(βgal)]}), and the ratio for GFP-transfected cells was set as 1. Bars, mean ± SD of three independent experiments.

Figure 4.

Cytoplasmic localization of hnRNP A1 reduces translation of endogenous XIAP mRNA. (A) Overexpression of hnRNP A1 reduces endogenous XIAP levels. HEK293T cells were transfected with a plasmid expressing GFP, FLAG-hnRNP A1, or FLAG-hnRNP A1 F1 mutant. Forty-eight hours after transfection protein extracts were prepared, separated by SDS-PAGE, transferred to PVDF membrane, and subjected to Western blot analysis with antibodies against XIAP and GAPDH. Relative XIAP levels are expressed as a ratio of XIAP to GAPDH. Displayed data are representative of three independent experiments. (B) Overexpression of the cytoplasmic hnRNP A1 F1 mutant does not affect total protein synthesis. HEK293T cells were transfected with a plasmid expressing GFP or the FLAG-hnRNP A1 F1 mutant, and 24 h later cells were metabolically labeled in the presence of [³⁵S]methionine for 20 min. Protein extracts were harvested, and equal amounts were separated by SDS-PAGE. Proteins were visualized by Coomassie Brilliant Blue staining and autoradiography. (C) Overexpression of hnRNP A1 does not affect XIAP mRNA levels. Cells were treated as described in A; total RNA was isolated 48 h after transfection, and cDNA was generated by reverse transcription. Quantitative PCR was used to determine the levels of XIAP and GAPDH mRNAs; values are expressed as XIAP relative to GAPDH (2^{−[Ct(XIAP) − Ct(GAPDH)]}), and the ratio for GFP-transfected cells was set as 1. Bars, mean ± SD of three experiments. (D) Overexpression of the cytoplasmic hnRNP A1 F1 mutant does not affect XIAP protein stability. Cells were transfected with plasmids expressing GFP or the FLAG-hnRNP A1 F1 mutant. Eighteen hours after transfection cells were treated with 10 μg/ml cyloheximide for 45 min, and then samples were harvested at 0-, 1-, 2-, 4-, and 8-h time points. Equivalent amounts of protein extract were separated by SDS-PAGE, transferred to PVDF, and analyzed by Western blot using antibodies against XIAP and GAPDH. The graph shows XIAP protein levels relative to GAPDH levels (bars), with the 0-h time point set as 100.

Figure 5.

Stress-induced cytoplasmic accumulation of hnRNP A1 reduces XIAP protein levels. (A) Osmotic shock redistributes hnRNP A1 to the cytoplasm and reduces XIAP protein levels. HEK293T cells were grown in high-osmolarity growth medium (0.6 M OSM) or DMEM for 4 h, and nuclear (NE) and cytoplasmic (CE) protein fractions were harvested at 1-h intervals, separated by SDS-PAGE, transferred to PVDF, and subjected to Western blot analysis with antibodies against XIAP, hnRNP A1, and GAPDH. (B) Osmotic shock does not affect XIAP mRNA levels. HEK293T cells were treated with DMEM (Control) or 0.6 M OSM for 4 h, total RNA was then isolated and cDNA was generated by reverse transcription. Quantitative PCR was used to determine the levels of XIAP and GAPDH mRNAs; values are expressed as XIAP relative to GAPDH (2^{−[Ct(XIAP) − Ct(GAPDH)]}), and the ratio for control cells was set as 1. Bars, mean ± SD of three experiments. (C) Reduced XIAP expression during osmotic shock is dependent on hnRNP A1. HEK293T cells were transfected with 20 nM hnRNP A1 siRNA or 20 nM nonsilencing siRNA (control); 72 h later the media was replaced with 0.6 M OSM or DMEM and cells were incubated for an additional 5 h. Protein extracts were prepared, separated by SDS-PAGE, transferred to PVDF membrane, and subjected to Western blot analysis with antibodies against XIAP, hnRNP A1, and GAPDH.