Heterogeneous nuclear ribonucleoprotein A1 regulates cyclin D1 and c-myc internal ribosome entry site function through Akt signaling

Abstract

The translation of the cyclin D1 and c-myc mRNAs occurs via internal ribosome entry site (IRES)-mediated initiation under conditions of reduced eIF-4F complex formation and Akt activity. Here we identify hnRNP A1 as an IRES trans-acting factor that regulates cyclin D1 and c-myc IRES activity, depending on the Akt status of the cell. hnRNP A1 binds both IRESs in vitro and in intact cells and enhances in vitro IRES-dependent reporter expression. Akt regulates this IRES activity by inducing phosphorylation of hnRNP A1 on serine 199. Serine 199-phosphorylated hnRNP A1 binds to the IRESs normally but is unable to support IRES activity in vitro. Reducing expression levels of hnRNP A1 or overexpressing a dominant negative version of the protein markedly inhibits rapamycin-stimulated IRES activity in cells and correlated with redistribution of cyclin D1 and c-myc transcripts from heavy polysomes to monosomes. Importantly, knockdown of hnRNP A1 also renders quiescent Akt-containing cells sensitive to rapamycin-induced G(1) arrest. These results support a role for hnRNP A1 in mediating rapamycin-induced alterations of cyclin D1 and c-myc IRES activity in an Akt-dependent manner and provide the first direct link between Akt and the regulation of IRES activity.

FIGURE 1.

The 5′-UTRs of the human cyclin D1 and c-myc mRNAs contain short regions that exhibit Akt-dependent IRES activity following rapamycin exposure. A, schematic diagram showing dicistronic constructs. B, DNA segments corresponding to the indicated regions of the 5′-UTRs of cyclin D1 and c-myc were inserted into the intercistronic region of the dicistronic plasmid pRF. PTEN^-/- and PTEN^+/+ MEFs were transfected with the indicated plasmids (2 μg each) and treated with or without rapamycin (rapa) (10 nm), and luciferase activities were determined. Relative firefly luciferase (IRES-mediated initiation) activity is shown in the absence (open bars) or presence (shaded bars) of rapamycin and normalized to values obtained for pRF in each cell line. The mean and S.D. are shown for three independent experiments.

FIGURE 2.

Binding of hnRNP A1 to minimal cyclin D1 and c-myc IRESs in a yeast three-hybrid system. A, schematic diagram showing components of the three-hybrid assay. Clones encoding hnRNP A1 fused to a transcriptional activation domain bound a bifunctional hybrid RNA composed of either the cyclin D1 or c-myc IRES fused to MS2 coat protein RNA binding sites. The hybrid RNA also bound a LexA DNA-binding domain fusion to the bacteriophage MS2 coat protein. This ternary interaction reconstituted a functional transcription factor that interacted with LexA operator sites and resulted in activation of HIS3 and lacZ reporter gene transcription. B, L40uraMS2 cells carrying pHybLex/Zeo-MS2 (LexA DNA-binding domain/MS2 coat protein fusion-expressing plasmid) were transformed with the indicated constructs. Transformants were grown on selective media lacking tryptophan, uracil, and histidine and containing 5 mm 3-aminotriazole and analyzed via a colony color assay for lacZ expression. pRH5′-D1IRES, plasmid expressing MS2-cyclin D1 IRES hybrid RNA; pRH5′-mycIRES, plasmid expressing MS2-myc IRES hybrid RNA; pRH5′-ASD1IRES, plasmid expressing MS2-cyclin D1 IRES in antisense orientation; pRH5′-ASmycIRES, plasmid expressing MS2-myc IRES in antisense orientation; pYesTrp2, plasmid expressing activation domain only; pYesTrp2-A1, plasmid expressing hnRNP A1-activation domain fusion. As a positive control, the interaction between the iron-responsive protein (IRP) and its RNA binding sites (IRE) was detected using the hybrid RNA-expressing plasmid, pRH3′-IRE, and the IRP-activation domain fusion protein expressing plasmid pYesTrp2-IRP.

FIGURE 3.

Association of hnRNP A1 with the cyclin D1 and c-myc IRESs in vitro and in intact cells. A, RNA-EMSA of ³²P-labeled cyclin D1 or c-myc minimal IRES sequences incubated with GST-control (-,-) or GST-hnRNP A1 (-,+). Supershift was assessed by the addition of anti-GST antibody (+,+). B, hnRNP A1 binding curves for the cyclin D1 (left) IRES RNA (165 nucleotides) and the c-myc (center) IRES RNA (233 nucleotides). Right, competition filter binding assays. hnRNP A1-cyclin D1 IRES RNA complexes competed for binding against unlabeled cyclin D1 IRES RNA (⋄) or unlabeled p27^Kip1 IRES RNA (▵). Similarly, hnRNP A1-c-myc IRES RNA complexes competed for binding against unlabeled c-myc IRES RNA (♦) or unlabeled p27^Kip1 IRES RNA (▾). C, hnRNP A1 binds cyclin D1 IRES and c-myc IRES in cells. Control IgG or anti-hnRNP A1 antibody was used to immunoprecipitate (IP) lysates from the indicated cell lines, and bound RNA was amplified by PCR of the cyclin D1 (left) or c-myc (right) IRES sequences. PCR products were visualized via ethidium bromide staining. SM, DNA size markers. D, identification of hnRNP A1 in RNA pull-down assays utilizing biotinylated cyclin D1 or c-myc IRES RNAs. Cytoplasmic extracts of PTEN^-/- or PTEN^+/+ MEFs, which had been treated with or without rapamycin (10 nm), were incubated with biotinylated cyclin D1 or c-myc IRES RNAs or control RNAs corresponding to the antisense cyclin D1 or c-myc IRES sequences and precipitated with streptavidin-Sepharose beads. Input and bound fractions were analyzed by immunoblotting for hnRNP A1.

FIGURE 4.

Akt phosphorylates hnRNP A1 in vitro and in cells. A, schematic diagram of GST fusion proteins used for in vitro and in vivo Akt phosphorylation experiments. B, 500 ng of either wild type (wt) hnRNP A1 or A1-S199A was incubated with 200 ng of activated Akt and immunoblotted with anti-phospho-Akt substrate antibody, anti-GST antibody, or anti-Akt antibody. Phosphorylation reactions were performed for 0 min (negative control) and 30 min. C, hnRNP A1 phosphorylation in cells stimulated by serum. 293 cells transiently transfected for 24 h with 1 mg of each of the indicated constructs were deprived of phosphate and serum for 1 h, incubated in the absence or presence of LY294002 (20 mm) or wortmannin (100 nm) for 1 h, and exposed to [³²P]orthophosphate and dialyzed serum for 2 h. Immunoprecipitated hnRNP A1 was assessed for ³²P-labeling and protein levels with anti-GST antibody. The data are shown as mean densitometric ratios (³²P labeling/protein) from three independent experiments. D, Akt phosphorylates hnRNP A1 on serine 199 in serum-stimulated 293 cells. Cells were transfected with the indicated constructs and were deprived of phosphate and serum for 1 h and exposed to [³²P]orthophosphate and dialyzed serum as in C. Data are representative of three independent experiments. IP, immunoprecipitation; W, Western blot. E, endogenous association of Akt and hnRNP A1 in 293 cells. Cells were serum-starved for 24 h and then treated with or without dialyzed serum for 2 h. Endogenous Akt (left) or hnRNP A1 (right) was immunoprecipitated (IP) from extracts and immunoblotted for the indicated proteins. Aktp, immunoprecipitation with anti-Akt antibody preincubated with an Akt-blocking peptide. Three independent experiments were performed with similar results.

FIGURE 5.

Serine 199 is differentially phosphorylated in cyclin D1 or c-myc IRES bound hnRNP A1 in an Akt-dependent manner following rapamycin exposure. Biotinylated cyclin D1 or c-myc IRES RNAs were used to pull-down hnRNP A1 from cytoplasmic extracts of PTEN^-/- or PTEN^+/+ MEFs treated with or without rapamycin (10 nm) for 2.5 h. RNA-protein complexes were resolved by gel electrophoresis and immunoblotted with anti-hnRNP A1, anti-phospho-Akt substrate or serine 199 phospho-specific hnRNP A1 antibodies. Data are representative of three independent experiments.

FIGURE 6.

Akt negatively regulates cyclin D1 and c-myc IRES activity in vitro. A, siRNA mediated knockdown of hnRNP A1. U87 and U87_PTEN were transiently transfected with siRNAs targeting hnRNP A1 or a nontargeting scrambled (scr) sequence and exposed to rapamycin (10 nm) for 24 h, and extracts were prepared and immunoblotted for hnRNP A1 and actin as indicated. Similar results were found in two additional independent experiments. B, translation-competent extracts were prepared from U87_PTEN cells in which hnRNP A1 expression was knocked down via siRNA treatment, and the indicated proteins were added to the extracts prior to programming the extracts with either in vitro transcribed dicistronic cyclin D1 (B, left) or dicistronic c-myc (B, right) reporter mRNAs. Translations were performed at 30 °C for 40 min. An irrelevant ITAF, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was added as a negative control. Renilla (shaded bars) and firefly (open bars) luciferase activities were determined and normalized to values obtained for extracts alone. The mean and S.D. are shown for three independent experiments.

FIGURE 7.

Knockdown of hnRNP A1 abrogates Akt-dependent cyclin D1 and c-myc IRES activity following rapamycin exposure. U87 and U87_PTEN cells in which hnRNP A1 expression was inhibited via siRNA were transiently transfected with the indicated dicistronic reporter constructs as in Fig. 1. Relative firefly luciferase (IRES-mediated initiation) activity is shown in the absence (open bars) or presence (shaded bars) of rapamycin and normalized to values obtained for pRF in each cell line. The mean and S.D. are shown for three independent experiments.

FIGURE 8.

Effects of a dominant negative shuttling-deficient hnRNP A1 mutant on Akt-dependent cyclin D1 and c-myc IRES activity following rapamycin exposure. A, expression of the NLS-A1-HA mutant in U87 cells. Immunofluorescence microscopy of untransduced (mock-infected, top row) or NLS-A1-HA-transduced (bottom row) cells after dual immunofluorescence staining using anti-HA (red) and anti-hnRNP A1 (green) antibodies; the panels on the right shows the overlay composite images. B, U87 and U87_PTEN cells were stably transduced with the indicated viral constructs and transiently transfected with the indicated dicistronic reporter constructs as before. Relative firefly luciferase (IRES-mediated initiation) activity is shown in the absence (open bars) or presence (shaded bars) of rapamycin and normalized to values obtained for pRF in each cell line. The mean and S.D. are shown for three independent experiments.

FIGURE 9.

Knockdown of hnRNP A1 alters polysome distribution of cyclin D1 and c-myc mRNAs. A, polysome distributions of cyclin D1, c-myc, and actin mRNAs in hnRNP A1 knockdown PTEN^-/- and PTEN^+/+ MEFs treated with or without rapamycin. Extracts were prepared from PTEN^-/- or PTEN^+/+ MEFs transfected with the indicated siRNAs (control, nontargeting scrambled siRNA (scr) or hnRNP A1-targeting siRNA) and treated with or without rapamycin (10 nm) for 24 h. Extracts were subjected to sucrose density gradient centrifugation and then divided into 11 1-ml fractions, which were pooled into a nonribosomal, monosomal fraction (N, white bars) and a polysomal fraction (P, black bars). Purified RNAs were used in real time quantitative RT-PCR analysis to determine the distributions of actin, cyclin D1, and c-myc mRNAs across the gradients. Polysome tracings are shown above values obtained from the RT-PCR analyses, which are displayed graphically below. RT-PCR measurements were done in quadruplicate, and the mean and S.D. are shown. B, cyclin D1, c-myc, and actin protein levels in hnRNP A1 knockdown PTEN^-/- or PTEN^+/+ MEFs treated with or without rapamycin and transfected with the indicated siRNAs as in A. Experiments were repeated three times with similar results.

FIGURE 10.

Knockdown of hnRNP A1 confers sensitivity to quiescent Akt containing rapamycin resistant cells. A, U87 or U87_PTEN cells were transfected with the indicated siRNAs and treated with or without rapamycin (100 nm) for 48 h. S-phase cell cycle distribution was subsequently determined on propidium iodide-stained cells by flow cytometry. The mean and S.D. are shown for three independent experiments (^*, p < 0.05). B, in cells containing active Akt, hnRNP A1 is differentially phosphorylated at serine 199, which inactivates IRES-mediated translation of cyclin D1 and c-myc mRNAs. The absence of this phosphorylation event results in hnRNP A1-mediated IRES activity in the face of rapamycin exposure.