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Nucleolin Discriminates Drastically between Long-Loop and Short-Loop Quadruplexes

Abstract : We investigate herein the interaction between nucleolin (NCL) and a set of G4 sequences derived from the CEB25 human minisatellite that adopt a parallel topology while differing in the length of the central loop (from nine nucleotides to one nucleotide). It is revealed that NCL strongly binds to long- loop (five to nine nucleotides) G4 while interacting weakly with the shorter variants (loop with fewer than three nucleotides). Photo-cross-linking experiments using 5-bromo-2′-deoxyuridine (BrU)-modified sequences further confirmed the loop-length dependency, thereby indicating that the WT-CEB25-L191 (nine- nucleotide loop) is the best G4 substrate. Quantitative proteomic analysis (LC-MS/MS) of the product(s) obtained by photo-cross- linking NCL to this sequence enabled the identification of one contact site corresponding to a 15-amino acid fragment located in helix α2 of RNA binding domain 2 (RBD2), which sheds light on the role of this structural element in G4-loop recognition. Then, the ability of a panel of benchmark G4 ligands to prevent the NCL−G4 interaction was explored. It was found that only the most potent ligand PhenDC3 can inhibit NCL binding, thereby suggesting that the terminal guanine quartet is also a strong determinant of G4 recognition, putatively through interaction with the RGG domain. This study describes the molecular mechanism by which NCL recognizes G4-containing long loops and leads to the proposal of a model implying a concerted action of RBD2 and RGG domains to achieve specific G4 recognition via a dual loop−quartet interaction.
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https://hal.archives-ouvertes.fr/hal-02999308
Contributor : Sophie Bombard <>
Submitted on : Tuesday, November 24, 2020 - 3:15:24 PM
Last modification on : Monday, November 30, 2020 - 3:51:48 PM

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Abhijit Saha, Patricia Duchambon, Vanessa Masson, Damarys Loew, Sophie Bombard, et al.. Nucleolin Discriminates Drastically between Long-Loop and Short-Loop Quadruplexes. Biochemistry, American Chemical Society, 2020, 59 (12), pp.1261-1272. ⟨10.1021/acs.biochem.9b01094⟩. ⟨hal-02999308⟩

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