Importin 7 and exportin 1 link c-Myc and p53 to regulation of ribosomal biogenesis

Abstract

Members of the β-karyopherin family mediate nuclear import of ribosomal proteins and export of ribosomal subunits, both required for ribosome biogenesis. We report that transcription of the β-karyopherin genes importin 7 (IPO7) and exportin 1 (XPO1), and several additional nuclear import receptors, is regulated positively by c-Myc and negatively by p53. Partial IPO7 depletion triggers p53 activation and p53-dependent growth arrest. Activation of p53 by IPO7 knockdown has distinct features of ribosomal biogenesis stress, with increased binding of Mdm2 to ribosomal proteins L5 and L11 (RPL5 and RPL11). Furthermore, p53 activation is dependent on RPL5 and RPL11. Of note, IPO7 and XPO1 are frequently overexpressed in cancer. Altogether, we propose that c-Myc and p53 counter each other in the regulation of elements within the nuclear transport machinery, thereby exerting opposing effects on the rate of ribosome biogenesis. Perturbation of this balance may play a significant role in promoting cancer.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Fig 1. IPO7 is a direct target of c-Myc

(A) HCT116 cells were transfected with siRNA (20 nM) targeted against c-Myc (sic-Myc) or control siRNA (siControl). Cells were harvested 48 hours later, and relative levels of IPO7 and XPO1 mRNA were measured using RT-qPCR. Levels of IPO7 (upper panel) and XPO1 (lower panel) mRNA were normalized to GAPDH mRNA in the same sample. Results are presented as fold change relative to non-treated siControl cells, taken as 1.0. The mean of 4 independent experiments is presented; bars indicate SD (*=p<0.05). (B) HCT116 and HT-29 cells were transfected and harvested as in (A), followed by SDS-PAGE and Western blot analysis for IPO7 and c-Myc proteins. GAPDH served as loading control. (C) Mouse embryonic fibroblasts (MEF) were infected with recombinant retroviruses encoding either GFP or c-Myc. 72 hours later, cells were harvested and relative levels of IPO7 and XPO1 mRNA were measured by qRT-PCR and normalized for GAPDH mRNA. Results are presented as fold change relative to the levels in GFP infected cells, taken as 1.0. Bars indicate SD (n=3, *=p<0.05). (D) MEF were infected as in (C). IPO7 protein levels were analyzed by Western blotting. (E) HCT116 and HT-29 cells were subjected to chromatin immunoprecipitation (ChIP) analysis with c-Myc antibody or control HA antibody. Primers flanking the putative c-Myc binding site were employed to amplify that region by qPCR. Primers located 3Kb upstream to the transcription start site were used as negative control. ChIP values are presented as percentage of the input. (F) ChIP was performed as in E, but with qPCR primers spanning the putative c-Myc site within the XPO1 first intron.

Fig 2. Reduced nuclear import of RPL4 in vivo following IPO7 depletion

(A) Nuclei of H1299 cells endogenously expressing YFP-tagged RPL4 were photobleached until less than 5% of the original signal remained detectable. Time lapse microscopy was employed to monitor nucleolar fluorescence signal recovery in cells transiently transfected with 20nM control siRNA or IPO7 siRNA. Pictures were taken at 5 minute time intervals. (B) The average fluorescence intensity within the nucleus of the bleached cells was measured. Each time point is expressed as the relative measured intensity divided by the pre-bleached signal. All values were corrected to the background signal. The average of 10 different cells is presented. Bars indicate SD. (C) Western blot analysis of IPO7, RPL4-YFP and GAPDH as loading control, in cell cultures corresponding to (A).

Fig 3. IPO7 knockdown causes altered nucleolar morphology

HCT116 cells were transfected with control or IPO7 siRNA (20 nM). 96 hours later, cells were fixed and immunostained with antibodies against fibrillarin (Green) and UBF (Red). Nuclei were counterstained with DAPI (Blue). Insets depict a higher magnification of the cell indicated by the small square. Scale bar (bottom right panel) = 10 μm.

Fig 4. IPO7 knockdown triggers p53 activation and growth inhibition

(A) HCT116 cells (wt and p53-null) were transiently transfected with IPO7 siRNA (20nM), control siRNA (20nM) or a combination of both siRNAs (20nM each). 96 hours later cells were trypsinized and re-plated at 5×103 cells/well in a 6 well dish. After an additional 7 days, colonies were stained with crystal violet. (B) MRC5 and HCT116 cells were transiently transfected with the indicated siRNA oligonucleotides (20nM). 96 hours later cells were harvested and subjected to Western blot analysis with antibodies against IPO7, p53, p21 and GAPDH as a loading control. (C, D) qRT-PCR analysis of p21 mRNA levels in MRC5 (left) and wild type (p53+/+) and p53-deficient (p53−/−) HCT116 cells (right) 96 hours after transient transfection with the indicated combinations of siRNA oligonucleotides (20nM each). p21 was normalized to GAPDH mRNA. Results are presented as fold change relative to non-treated siControl cells, taken as 1.0. Bars indicate SD; n=3 (** =p<0.01).

Fig 5. Activation of p53 following IPO7 knockdown is mediated through RPL11 and RPL5

(A) HCT116 cells were transiently transfected with the indicated combinations of siRNA targeting IPO7 or RPL11 (20nM each). 72 hours later cells were harvested and protein extracts subjected to Western blot analysis with the indicated antibodies. GAPDH served as a loading control. (B) HCT116 were transiently transfected as in (A) except that cells were transfected with RPL5 siRNA. (C) RPL11 associates with Mdm2 in cells depleted of IPO7. HCT116 cells were transiently transfected with siIPO7 or siControl (20nM) and harvested 72 hours later. Proteins were extracted in NP-40 lysis buffer, and incubated with anti-Mdm2 antibodies or anti-HA tag antibodies as negative control. Immunoprecipitated proteins (IP, right panel) as well as 5% of each total cell extract (Input, left panel) were subjected to Western blot analysis with the indicated antibodies.

Fig 6. IPO7 gene transcription is repressed by p53

(A,B) MRC5 cells were transfected with p53 siRNA (sip53) or control siRNA. IPO7 (A) and XPO1 (B) mRNA levels were measured using qRT-PCR and normalized for the corresponding GAPDH mRNA. Results are presented as fold change relative to non-treated siControl cells, taken as 1.0. Error bars indicate SD (n=3). (* = p<0.05). (C) Wild type (WT) and p53 knockout (KO) mouse embryonic fibroblasts (MEF) were infected with recombinant retroviruses expressing c-Myc or GFP as control. 72 hours post infection, cells were harvested and relative levels of IPO7 mRNA determined as in (A). Results are presented as fold change relative to the levels in the GFP infected cells, taken as 1.0. Error bars represent standard deviation (n=3). (D) Western blot analysis of IPO7 protein levels in cultures treated as in (C). GAPDH served as loading control. (E) XPO1 mRNA was quantified as in (C). Error bars represent standard deviation (n=3). (F) MRC5 cells were transfected with p53 siRNA (sip53) or control siRNA. 72 hours later, cells were treated with solvent (ethanol) only (NT) or 5nM actinomycin D (ActD) in ethanol for an additional 24 hours. Levels of mature IPO7 and XPO1 mRNA, as well as of the corresponding heterogeneous nuclear RNA, representing primary transcripts and identified through the use of intronic primers (Intronic), were measured by qRT-PCR and normalized for GAPDH mRNA. Results are presented as fold change relative to non-treated siControl cells, taken as 1.0. Error bars indicate SD (n=3). Right panel: Western blot analysis of p53 protein levels in the same samples.

Figure 7. p53 associates with the IPO7 and XPO1 genes

(A) MRC5 cells were treated for 16 hours with 5nM Actinomycin D (ActD) or solvent only (NT), and then subjected to chromatin immunoprecipitation (ChIP) analysis with p53-specific antibody (p53) or control HA antibody. Primers flanking the putative p53 binding sites were employed to amplify the corresponding DNA regions by RT-qPCR. For the IPO7 gene, two different sites were amplified, termed site 1 and site 2, in the IPO7 promoter and first intron, respectively. Primers derived from the GAPDH gene served as negative control. ChIP values are presented as percentage of input. Bars represent standard deviation. (B) Same as in (A), except that the analysis was done with XPO1 primers. (C) Schematic model depicting the opposing regulation of the nuclear import/export machinery by c-Myc and p53. Positive and negative regulatory interactions are indicated. RP = ribosomal proteins; 60S and 40S refer to the corresponding ribosomal subunits.