HuR recruits let-7/RISC to repress c-Myc expression

Abstract

RNA-binding proteins (RBPs) and microRNAs (miRNAs) are potent post-transcriptional regulators of gene expression. Here, we show that the RBP HuR reduced c-Myc expression by associating with the c-Myc 3' untranslated region (UTR) next to a miRNA let-7-binding site. Lowering HuR or let-7 levels relieved the translational repression of c-Myc. Unexpectedly, HuR and let-7 repressed c-Myc through an interdependent mechanism, as let-7 required HuR to reduce c-Myc expression and HuR required let-7 to inhibit c-Myc expression. Our findings suggest a regulatory paradigm wherein HuR inhibits c-Myc expression by recruiting let-7-loaded RISC (RNA miRNA-induced silencing complex) to the c-Myc 3'UTR.

Figure 1.

HuR represses c-Myc expression. (A) HeLa cell lysates were subjected to RNP IP followed by RT–qPCR analysis to measure the enrichment of c-Myc mRNA in HuR IP compared with control IgG IP (Materials and Methods). (B) Forty-eight hours after transfection of HeLa cells with control (Ctrl) siRNA or HuR-directed siRNA, lysates were prepared to assess the levels of c-Myc, HuR, and loading control GAPDH by Western blot analysis (top) and the levels of c-Myc mRNA by RT–qPCR, using 18S rRNA for normalization (bottom). (C) Forty-eight hours after transfection with a control plasmid expressing GFP or a plasmid overexpressing HuR as fusion protein GFP-HuR, the levels of c-Myc, GFP-HuR, endogenous HuR, and GAPDH (top), and c-Myc mRNA (bottom) were measured as explained in B. (D) Plasmid pGFP-3′UTR was constructed by attaching the entire c-Myc 3′UTR after the GFP CR. (E) By 48 h after transfecting pGFP or pGFP-3′UTR, the levels of reporter GFP protein, HuR, and loading control β-actin (left) and GFP and chimeric GFP-3′UTR mRNAs (right) were measured as explained in B. (F) Sequence of the AU-rich c-Myc 3′UTR and schematic depiction of the 5′UTR, CR (CR1, CR2), and 3′UTR (A–D) biotinylated RNAs (assayed in duplicate) used for biotin pull-down analysis (shown in G) (Materials and Methods). (Input) Positive control; (biotinylated GAPDH 3′UTR) negative control. HuR in biotin pull-down samples was detected by Western blot analysis. (H) Constructs were prepared to express chimeric RNAs spanning the GFP CR and each of the four c-Myc 3′UTR segments shown in F; 48 h after transfection, binding of HuR to each chimeric RNA was tested by RNP IP followed by GFP mRNA detection by RT–qPCR. Values in A–C and E are the means ± SD from three independent experiments. The Western blotting data are representative of three or more experiments. Where indicated, c-Myc Western blotting signals were quantified by densitometry.

Figure 2.

let-7b/c inhibits c-Myc expression in an HuR-dependent fashion. (A) Schematic of the let-7 interaction site on the c-Myc 3′UTR (hatched). (B) Forty-eight hours after transfection of (AS)let-7b/c or (Pre)let-7b/c, the levels of c-Myc, HuR, and loading control GAPDH (left) were tested by Western blot analysis, and the levels of c-Myc mRNA (right) were measured by RT–qPCR. (C) Forty-eight hours after transfection of Ctrl or HuR siRNAs, the levels of let-7b and let-7c were measured by RT–qPCR analysis. (D) In cells transfected as described in B, the interaction of HuR with c-Myc mRNA was measured by RNP IP followed by RT–qPCR analysis. (E) To test the influence of let-7b/c on c-Myc expression, GFP reporters were prepared in which GFP was linked to the AB segment containing the intact let-7 site (GFP-AB) or a mutant let-7 site (GFP-ABmut) with four point mutations (*) in the seed region that disrupted the interaction of let-7b and let-7c with the c-Myc mRNA (gray highlight). (F,G) Thirty-six hours after transfection of Ctrl siRNA or (AS)let-7b/c (F) or (Pre)let/7b/c (G), plasmid pGFP, pGFP-AB or pGFP-ABmut were transfected and the levels of GFP and loading control β-actin tested 24 h later by Western blot analysis. (H) Following transfection of Ctrl or HuR siRNAs together with plasmids pGFP, pGFP-AB or pGFP-ABmut as explained in F and G, the levels of GFP, HuR, and β-actin were assessed by Western blot analysis (left) and quantified (right). Values in B, C, and H are the means ± SD from three independent experiments. Western blotting data are representative of three or more experiments. Where indicated, “(fold),” and in H, c-Myc or GFP Western blotting signals were quantified by densitometry.

Figure 3.

Interaction of c-Myc mRNA with Ago2 requires HuR and is enhanced by let-7b/c. (A) Forty-eight hours after silencing Ago1 or Ago2 by using siRNAs (Supplemental Fig. S5), the levels of c-Myc protein was evaluated by Western blot analysis (left) and the levels of c-Myc mRNA by RT–qPCR analysis (right). (B) Twenty-four hours after transfecting HeLa cells with Ago1, Ago2, and Ctrl siRNAs, cells were further transfected with Ctrl siRNA or (Pre)let-7b/c for an additional 24 h, whereupon the levels of c-Myc were determined by Western blot analysis. (C) The interaction of Ago2 with HuR was tested by IP with IgG or anti-Ago2 antibodies, followed by HuR detection by Western blot analysis in lysates that were either left untreated or digested with RNase A. (D–F) Forty-eight hours after transfection with the indicated RNAs (D,E) and plasmids (F), the levels of c-Myc mRNA present in Ago2 IP were measured by RT–qPCR analysis. (G) Cells were transfected as described in B, except that the influence of (Pre)let-7b/c on c-Myc mRNA interaction with Ago2 was tested in cells with normal (Ctrl siRNA) and reduced (HuR siRNA) HuR levels. (H) Plasmids pAB-MS2 [pcDNA-MS2(12X)-AB] and pMS2 [pcDNA-MS2(12X)] were derived from plasmid pSL-MS2(12X) (Fusco et al. 2003) and the levels of let-7b and let-7c were measured by RT–qPCR in cells expressing either normal (Ctrl siRNA) or reduced (HuR siRNA) HuR by 48 h after transfection (graph). (I) Forty-eight hours after transfection of cells with either Ctrl or HuR siRNAs, in the absence or presence of (Pre)let-7b/c, the levels of c-Myc, HuR, and β-actin were tested by Western blot analysis; c-Myc levels were quantified by densitometry. Values in A and D–H are the means ± SD from three independent experiments. The Western blotting data are representative of three or more experiments.

Figure 4.

Proposed model of HuR repression of c-Myc by recruiting let-7/RISC to c-Myc 3′UTR. (A) The presence of elevated HuR promotes the association of let-7-loaded RISC on the c-Myc 3′UTR, in turn lowering c-Myc mRNA levels and translation. (B) In the presence of low HuR levels, let-7-loaded RISC has lower affinity for the c-Myc 3′UTR, and c-Myc production is enhanced. See the text for details.