Identification and Characterization of Nucleolin as a c-myc G-quadruplex-binding Protein

Abstract

myc is a proto-oncogene that plays an important role in the promotion of cellular growth and proliferation. Understanding the regulation of c-myc is important in cancer biology, as it is overexpressed in a wide variety of human cancers, including most gynecological, breast, and colon cancers. We previously demonstrated that a guanine-rich region upstream of the P1 promoter of c-myc that controls 85-90% of the transcriptional activation of this gene can form an intramolecular G-quadruplex (G4) that functions as a transcriptional repressor element. In this study, we used an affinity column to purify proteins that selectively bind to the human c-myc G-quadruplex. We found that nucleolin, a multifunctional phosphoprotein, binds in vitro to the c-myc G-quadruplex structure with high affinity and selectivity when compared with other known quadruplex structures. In addition, we demonstrate that upon binding, nucleolin facilitates the formation and increases the stability of the c-myc G-quadruplex structure. Furthermore, we provide evidence that nucleolin overexpression reduces the activity of a c-myc promoter in plasmid presumably by inducing and stabilizing the formation of the c-myc G-quadruplex. Finally, we show that nucleolin binds to the c-myc promoter in HeLa cells, which indicates that this interaction occurs in vivo. In summary, nucleolin may induce c-myc G4 formation in vivo.

Translational Repression of the Disintegrin and Metalloprotease ADAM10 by a Stable G-quadruplex Secondary Structure in Its 5′-Untranslated Region

“…Deficiencies of certain G-quadruplex resolving helicases result in abnormal mRNA deadenylation and decay and in perturbed telomere maintenance and cellular DNA replication, leading to cancer (87). In addition, it was reported recently that nucleolin stabilizes the G-quadruplex within the c-myc promoter and inhibits transcription (88). Interestingly, nucleolin interacts with G-rich sequences in UTRs or the coding region of a lot of mRNAs and was found to enhance their translation (89), suggesting that nucleolin might function as a RNA-G-quadruplex interacting factor.…”

The Requirement of c-Jun N-terminal Kinase 2 in Regulation of Hypoxia-inducing Factor-1α mRNA Stability

“…Previous work revealed that nucleolin had high affinity to G-quadruplex structure (27), including the sequences in the 5Ј-UTR region of hif-1␣ mRNA. The study reported here provided in vivo evidence for the physical interaction between nucleolin and hif-1␣ mRNA using RNA-IP assay with nucleolin antibody.…”

“…Nucleolin has been found to bind to the mRNA of several important genes, including p53 (24), bcl-2 (25), and bcl-xl (26), leading to regulation on mRNA turnover or translation. Moreover, nucleolin has been shown to have binding activity to a number of G-rich oligonucleotides, like the G-quadruplex structure in the promoter region of c-myc gene (27) and vegf gene (28). Gonzalea et al showed, in an in vitro filter binding assay, that nucleolin bound to the G-quadruplex structure in 5Ј-UTR of hif-1␣ mRNA (27).…”

“…Moreover, nucleolin has been shown to have binding activity to a number of G-rich oligonucleotides, like the G-quadruplex structure in the promoter region of c-myc gene (27) and vegf gene (28). Gonzalea et al showed, in an in vitro filter binding assay, that nucleolin bound to the G-quadruplex structure in 5Ј-UTR of hif-1␣ mRNA (27). However, it is still open for investigation as to whether or not nucleolin could regulate HIF-1␣ expression in vivo.…”

Nucleolin and nucleophosmin: nucleolar proteins with multiple functions in DNA repair

“…For D r a f t 13 example, NCL has been reported to cooperate with hnRNP D to form the heterodimeric LR1 Gquadruplex-binding complex that transcriptionally activates IgH sequences in B-cells (Dempsey et al 1998;Hanakahi et al 1997). In contrast, NCL binding to and stabilization of a defined G-quadruplex structure within the c-MYC gene promoter contributes to transcriptional repression of this locus, possibly by abolishing binding sites for activating TFs or altering the local chromatin (González et al 2009;González and Hurley 2010). Post-transcriptionally, NCL binding to mRNAs may effect expression of particular proteins, with NCL binding to the p53 mRNA 5'UTR resulting in translational activation, while its binding to gadd45α mRNA in response to arsenite treatment and K294-SUMOylation blocks the degradation of this transcript (Takagi et al 2005;Zhang et al 2015).…”