Abstract
BackgroundG-quadruplexes (G4s) are four-stranded nucleic acid structures that form in G-rich sequences. Nucleolin (NCL) is a cellular protein reported for its functions upon G4 recognition, such as induction of neurodegenerative diseases, tumor and virus mechanisms activation. We here aimed at defining NCL/G4 binding determinants. MethodsElectrophoresis mobility shift assay was used to detect NCL/G4 binding; circular dichroism to assess G4 folding, topology and stability; dimethylsulfate footprinting to detect G bases involved in G4 folding. ResultsThe purified full-length human NCL was initially tested on telomeric G4 target sequences to allow for modulation of loop, conformation, length, G-tract number, stability. G4s in promoter regions with more complex sequences were next employed. We found that NCL binding to G4s heavily relies on G4 loop length, independently of the conformation and oligonucleotide/loop sequence. Low stability G4s are preferred. When alternative G4 conformations are possible, those with longer loops are preferred upon binding to NCL, even if G-tracts need to be spared from G4 folding. ConclusionsOur data provide insight into how G4s and the associated proteins may control the ON/OFF molecular switch to several pathological processes, including neurodegeneration, tumor and virus activation. Understanding these regulatory determinants is the first step towards the development of targeted therapies. General significanceThe indication that NCL binding preferentially stimulates and induces folding of G4s containing long loops suggests NCL ability to modify the overall structure and steric hindrance of the involved nucleic acid regions. This protein-induced modification of the G4 structure may represent a cellular mechanosensor mechanism to molecular signaling and disease pathogenesis. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.
Highlights
G-quadruplexes (G4s) are unique four-stranded nucleic acid structures that may form in guanine-rich sequences [1]
Our present work indicates that long loops in the G4 structure are preferred and this behavior is independent of the G4 conformation or loop/oligonucleotide sequence
These apparent differences may be explained by several reasons: 1) the use of a recombinant NCL purified in bacteria [41] vs the full-length human NCL purified from human cells: the recombinant protein included all RNA binding domains (RBD) and C-terminal domain of the natural human protein but lacked the N-terminal domain which in turn was fused to the Escherichia coli maltose-binding protein; even if the nucleic acid binding activity was maintained in these conditions, the specific recognition of G4s, and in particular of their steric features, may be severely distorted by the introduction of a different Nterminal domain
Summary
G-quadruplexes (G4s) are unique four-stranded nucleic acid structures that may form in guanine-rich sequences [1]. Conclusions: Our data provide insight into how G4s and the associated proteins may control the ON/OFF molecular switch to several pathological processes, including neurodegeneration, tumor and virus activation Understanding these regulatory determinants is the first step towards the development of targeted therapies. General significance: The indication that NCL binding preferentially stimulates and induces folding of G4s containing long loops suggests NCL ability to modify the overall structure and steric hindrance of the involved nucleic acid regions. This protein-induced modification of the G4 structure may represent a cellular mechanosensor mechanism to molecular signaling and disease pathogenesis. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr Concetta Giancola and Dr Daniela Montesarchio
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