Constrained G4 structures unveil topology specificity of known and new G4 binding proteins

S. Britton,Y. Couté,P. Calsou,A. Adrait,A. Pipier,T. Lavergne,D. Gomez,E. Defrancq,J. F. Riou,A. Devaux

doi:10.1038/s41598-021-92806-8

Abstract

G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases. An essential feature of G4 is their intrinsic polymorphic nature, which is characterized by the equilibrium between several conformations (also called topologies) and the presence of different types of loops with variable lengths. In cells, G4 functions rely on protein or enzymatic factors that recognize and promote or resolve these structures. In order to characterize new G4-dependent mechanisms, extensive researches aimed at identifying new G4 binding proteins. Using G-rich single-stranded oligonucleotides that adopt non-controlled G4 conformations, a large number of G4-binding proteins have been identified in vitro, but their specificity towards G4 topology remained unknown. Constrained G4 structures are biomolecular objects based on the use of a rigid cyclic peptide scaffold as a template for directing the intramolecular assembly of the anchored oligonucleotides into a single and stabilized G4 topology. Here, using various constrained RNA or DNA G4 as baits in human cell extracts, we establish the topology preference of several well-known G4-interacting factors. Moreover, we identify new G4-interacting proteins such as the NELF complex involved in the RNA-Pol II pausing mechanism, and we show that it impacts the clastogenic effect of the G4-ligand pyridostatin.

Highlights

G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases
The relationship between G4 structures/motifs and the progressive discovery of proteins that modulate the dynamic of G4 formation, such as helicases or other proteins involved in DNA and RNA transactions has expanded our knowledge to understand the ubiquitous function of G4 structures on cellular metabolism and cell fate[5,6,13,15,17,18,19,20,64]
We identified through a mass spectrometry (MS)-based quantitative proteomic analysis 425 human proteins significantly enriched on constrained G4 structures relative to a duplex DNA control, in which well-described G4 interacting factors were present

Summary

Introduction

G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases. Depending on the length and the composition of the sequence, as well as the environmental conditions (including the nature and concentration of metal cations, and local molecular crowding), a G4-forming sequence can adopt different topologies, in which the strands are in parallel, antiparallel or hybrid conformations, with the co-existence of different types of loops (lateral, diagonal or propeller) of variable lengths[9,10,11]. This polymorphism is exacerbated for the human telomeric sequence and leads to intricate structural mixtures[12]. The presence of G4 motifs in the TSS proximal regions is associated with RNA-Pol II pausing sites and R-loops formation, two different factors promoting RNA-Pol II arrests and transcription-dependent DNA b reaks[30,31,32,33,34]

Methods

Results

Conclusion