Ribosomal protein S14 negatively regulates c-Myc activity

Abstract

The ribosomal gene RPS14 is associated with the cancer-prone 5q-syndrome, which is caused by an interstitial deletion of the long arm of human chromosome 5. Previously, we found that ribosomal protein S14 (RPS14) binds to and inactivates MDM2, consequently leading to p53-dependent cell-cycle arrest and growth inhibition. However, it remains elusive whether RPS14 regulates cell proliferation in a p53-independent manner. Here, we show that RPS14 interacts with the Myc homology box II (MBII) and the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) domains of the oncoprotein c-Myc. Further, RPS14 inhibited c-Myc transcriptional activity by preventing the recruitment of c-Myc and its cofactor, TRRAP, to the target gene promoters, as thus suppressing c-Myc-induced cell proliferation. Also, siRNA-mediated RPS14 depletion elevated c-Myc transcriptional activity determined by its target gene, Nucleolin, expression. Interestingly, RPS14 depletion also resulted in the induction of c-Myc mRNA and subsequent protein levels. Consistent with this, RPS14 promoted c-Myc mRNA turnover through an Argonaute 2 (Ago2)- and microRNA-mediated pathway. Taken together, our study demonstrates that RPS14 negates c-Myc functions by directly inhibiting its transcriptional activity and mediating its mRNA degradation via miRNA.

Keywords: 5-q Syndrome; Oncogene; Proliferation; Ribosomal Protein L11; Ribosomal Protein S14; Signal Transduction; Transcription Regulation; c-Myc; p53.

FIGURE 1.

RPS14 associates with c-Myc at the MBII and bHLH-LZ domains. A and B, exogenous RPS14 binds to exogenous c-Myc in H1299 cells. Cells were transfected with V5-c-Myc and/or Flag-S14 plasmids as indicated and harvested at 48 h post-transfection for IP assays (0.3 mg total proteins per sample) using anti-Flag, anti-c-Myc (N262), or mouse/rabbit IgG, followed by IB assays with anti-c-Myc (N262) or anti-Flag. C and D, mapping the RPS14-binding domains of c-Myc. H1299 cells were transfected with Flag-S14 plasmid together with full-length c-Myc or c-Myc fragment-expressing plasmids and harvested at 48 h post-transfection for a set of IP assays using anti-V5 followed by IB assays with the antibodies as indicated. Arrows indicate the V5-c-Myc or V5-c-Myc-fragments. E, schematic representation of the RPS14-binding domains of c-Myc. F, endogenous RPS14 interacts with endogenous c-Myc in H1299 cells. Cell lysates (1 mg total proteins per sample) were prepared, and IP assays were performed using anti-c-Myc (N262) or rabbit IgG, followed by IB assays with anti-c-Myc (9E10) or anti-RPS14.

FIGURE 2.

RPS14 influences the recruitment of c-Myc and TRRAP to c-Myc target promoters. A, RPS14 overexpression reduces the recruitment of c-Myc and TRRAP to c-Myc target promoters. H1299 cells were transfected with control or Flag-S14 plasmid followed by serum starvation for overnight and stimulation for 3 h before harvesting for ChIP assay using antibodies or rabbit IgG as indicated. B, RPS14 overexpression reduces exogenous c-Myc recruitment to the target gene promoter. V5-c-Myc together with or without Flag-S14 was transfected into H1299 cells. Cells were harvested and subjected to ChIP assay using anti-c-Myc. C, siRNA-mediated knockdown of endogenous RPS14 increases the recruitment of c-Myc and TRRAP to the c-Myc target promoters. H1299 cells were transfected with control or RPS14 siRNAs for 48–72 h before harvesting for ChIP assays using antibodies or rabbit IgG and for IB (inset) as indicated. The co-immunoprecipitated DNAs in A, B, and C were subjected to qPCR analysis of the Nucleolin gene promoter. * indicates p < 0.05. D, RPS14 overexpression affects the c-Myc-Max interaction in H1299 cells. Cells were transfected with combinations of V5-c-Myc, pEGFP-Max, and Flag-S14 plasmids as indicated and were harvested for IP assays using anti-c-Myc (N262). The immunoprecipitated proteins were detected by IB assays with anti-V5, anti-GFP, or anti-Flag. E, RPS14 overexpression affects the interaction between the amino acids 355–439 of c-Myc and Max. The same experiments were performed as shown in D, except for using the V5-c-Myc/355–439 plasmid for transfection and anti-V5 for IP assays.

FIGURE 3.

RPS14 inhibits c-Myc transcriptional activity and subsequent cell proliferation in H1299 cells. A and B, overexpression of RPS14 down-regulates c-Myc transcriptional activity determined by E2F2 and Nucleolin expression. Cells were transfected with Flag-S14 and V5-c-Myc plasmids individually or together for 30 h and harvested for RT-qPCR analysis to detect the expression of c-Myc target genes, E2F2 and Nucleolin. C and D, RPS14 suppresses c-Myc-induced cell proliferation as determined by BrdU incorporation assays. Cells were transfected with Flag-S14 and V5-c-Myc plasmids individually or together for 30 h and supplemented with 10 μm BrdU for 2 to 3 h before subjected to BrdU staining (C). Percentage of BrdU-positive cells is shown in panel D. * indicates p < 0.05, ** indicates p < 0.005.

FIGURE 4.

Knockdown of RPS14 leads to the elevation of c-Myc activity and level. A and D, siRNA-mediated RPS14 depletion elevates the expression of Nucleolin in both H1299 and HCT116^p53−/− cells. Cells were transfected with control or RPS14 siRNAs for 48–72 h and supplemented with or without 5-FU overnight before harvesting for RT-qPCR. B and E, RPS14 knockdown increases c-Myc mRNA level as determined by RT-qPCR in both H1299 and HCT116^p53−/− cells. * indicates p < 0.05, ** indicates p < 0.005. C and F, the representative results for the expression of endogenous c-Myc and RPS14, which were detected by IB assays using antibodies as indicated.

FIGURE 5.

RPS14 interacts with Ago2 and leads to miR-145/Ago2-mediated c-Myc mRNA turnover. A and B, exogenous RPS14, but not RPL30, binds to exogenous Ago2 in H1299 cells. Cells were transfected with a combination of HA-Ago2, Flag-S14, and Flag-L30 plasmids as indicated and harvested at 48 h post-transfection for IP assays using anti-Flag, anti-HA, or mouse IgG, followed by IB assays with anti-HA or anti-Flag. C, the endogenous binding of RPS14 and Ago2 in H1299 cells. IP assays were performed using anti-Ago2 followed by IB assay using antibodies as indicated. D, Ago2 is required for suppression of c-Myc mRNA expression by RPS14. H1299 cells were transfected with plasmids or siRNA as indicated. Cells were harvested 48–72 h post-transfection and subjected to qPCR analysis (left panel) and IB assay (right panel). E, RPS14 knockdown impairs miR-145-mediated c-Myc mRNA decay. H1299 cells were transfected with miR-145 or siRNA as indicated. Cells were harvested 48–72 h post-transfection and subjected to qPCR analysis (left panel) and IB assay (right panel). * indicates p < 0.05. F, schematic for c-Myc regulation by ribosomal proteins via multiple mechanisms. Both RPS14 and RPL11 can negatively regulate c-Myc activity by preventing the recruitment of TRRAP to the c-Myc target promoters and prompting c-Myc mRNA degradation. Additionally, RPS14 can also hinder the binding of c-Myc to its target gene promoter.