The C-terminus of nucleolin promotes the formation of the c-MYC G-quadruplex and inhibits c-MYC promoter activity

Abstract

Nucleolin, the most abundant nucleolar phosphoprotein of eukaryotic cells, is known primarily for its role in ribosome biogenesis and cell proliferation. It is, however, a multifunctional protein that, depending on the cellular context, can drive either cell proliferation or apoptosis. Our laboratory recently demonstrated that nucleolin can function as a repressor of c-MYC transcription by binding to and stabilizing the formation of a G-quadruplex structure in a region of the c-MYC promoter responsible for controlling 85-90% of c-MYC's transcriptional activity. In this study, we investigate the structural elements of nucleolin that are required for c-MYC repression. The effect of nucleolin deletion mutants on the formation and stability of the c-MYC G-quadruplex, as well as c-MYC transcriptional activity, was assessed by circular dichroism spectropolarimetry, thermal stability, and in vitro transcription. Here we report that nucleolin's RNA binding domains 3 and 4, as well as the arginine-glycine-glycine (RGG) domain, are required to repress c-MYC transcription.

Fig. 1

Promoter structure of the c-MYC gene and scheme of its G-quadruplex structure. (A) Location of the NHE III₁ region within the c-MYC promoter. Runs of guanines that can participate in G-quadruplex formation are underlined. (B) Scheme of a guanine tetrad and a cartoon of the c-MYC G-quadruplex structure. Left: H-bonding pattern in a G-tetrad; center: schematic diagram of a G-tetrad; right: cartoon representing a G-quadruplex structure that is found in the c-MYC promoter region.

Fig. 2

Nucleolin deletion mutants. (A) Diagram of nucleolin structure. (B) Diagram of the nucleolin deletion mutants used in this study. Solid lines indicate regions of the nucleolin peptide that have been deleted from the Nuc-1,2,3,4-RGG construct. All proteins were overexpressed in E. coli fused at the N-terminal to the maltose-binding protein (MBP).

Fig. 3

Effect of deletion mutagenesis on the ability of nucleolin to induce c-MYC G-quadruplex formation. (A) CD spectra of Pu47ss after incubation with Nuc-1,2,3,4-RGG, or assay buffer containing 20 mM Tris-HCl, pH 7.4, 5 mM NaCl, 1 mM EDTA, in 50% glycerol. (B) CD spectra of Pu47ss after incubation with C-terminal nucleolin deletion mutants. (C) CD spectra of Pu47ss after incubation with nucleolin’s N-terminal deletion mutants. (D) Comparison of the effect of RBD-substitution on the ability of Nuc-3,4-RGG to induce G-quadruplex formation. Formation of a parallel G-quadruplex structure is reflected by the change in wavelength from 258 nm (single-stranded DNA) to 262 nm (G-quadruplex DNA) and increased molar ellipticity at 262 nm.

Fig. 4

Effect of deletion mutagenesis on the ability of nucleolin to promote the formation of a thermally stable c-MYC G-quadruplex. (A) Melting curves obtained for Pu47 containing the c-MYC G-quadruplex motif after incubation with the various nucleolin deletion mutants, including Nuc-1,2,3,4-RGG. (B) Effect of nucleolin C-terminal deletions on c-MYC G-quadruplex stability. (C) Effect of nucleolin N-terminal deletions on c-MYC G-quadruplex stability. (D) Comparison of the effects that Nuc-1,2-RGG, Nuc-2,3-RGG, and Nuc-3,4-RGG have on c-MYC G-quadruplex stabilization.

Fig. 5

Comparison of the binding affinity of Nuc-1,2,3,4-RGG, Nuc-3,4-RGG, and Nuc-RGG for the c-MYC G-quadruplex. 10,000 cpm of radiolabeled oligomer was assembled into a G-quadruplex structure and incubated with Nuc-1,2,3,4-RGG (A), Nuc-3,4-RGG (B), or Nuc-1,2,3,4-RGG (C) at the indicated concentrations in a 20-μL reaction containing 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM DTT, 50 ng/μL poly(dI-dC), 4 μg/mL BSA, and 25 mM KCl for 1 h at room temperature.

Fig. 6

Effect of Nuc-1,2,3,4-RGG, Nuc-3,4-RGG, and Nuc-RGG on c-MYC promoter activity as determined by in vitro transcription. (A) Diagram of the pMYC-Luc reporter construct containing the c-MYC NHE III₁ region that can assemble into a G-quadruplex structure. (B) Effect of Nuc-1,2,3,4-RGG (lanes 1–4), Nuc-3,4-RGG (lanes 5–8), and Nuc-RGG (lanes 9–12) on c-MYC promoter activity as determined by in vitro transcription assay. Reactions contained 1 μg of pMYC-WT (Del-4) plasmid and the indicated concentrations of nucleolin per reaction. Plasmid and the recombinant protein were incubated for 1 h at 4 °C prior to the addition of HeLa nuclear extract. In vitro transcription reactions were carried out at 42 °C for exactly 60 min, after which all reactions were stopped by the addition of stop buffer, as described in Materials and Methods. The multiple bands correspond to the transcripts from the several transcription initiation sites found in the luciferase coding region (44).