Chaperoning RNA and DNA G-quadruplexes that control proto-oncogenes expression.

PASCUTTI F.; HECKEL S.B; CHALLIER E; ARMAS P.; DAVID A.P.; BINOLFI A.

PRAGA

Congreso; G4thering - 6th International Meeting on Quadruplex Nucleic Acids.; 2017

G4thering - 6th International Meeting on Quadruplex Nucleic Acids.

G-quadruplexes (G4) have been found overrepresented not only in G-rich DNA in transcriptional control regions of human proto-oncogenes, but also in their RNA translational control regions. The fine-tuning of G4 formation in gene regulatory regions has emerged as a novel natural and artificial way to regulate gene expression. Here we report the activity of cellular nucleic acids binding protein (CNBP) on the formation of G4 relevant for expression regulation of proto-oncogenes. CNBP is a highly conserved vertebrates? protein with nucleic acids chaperone activity that has been implicated in transcriptional control through binding to ssDNA and translational control through binding to RNA and has been implicated in craniofacial development and in several human diseases (e.g. myotonic dystrophy type 2, sporadic inclusion body myositis and Treacher Collins syndrome). Here we analyzed CNBP binding and biochemical action on a parallel tetramolecular G4 formed by the DNA sequence TGGGGT using electrophoretic mobility shift assays (EMSA), circular dichroism (CD) spectroscopy, helicase-PAGE assays and 1D 1H-NMR, and we observed that CNBP has an ATP-independent G4-unfolding activity. Then we tested CNBP binding and G4-unwinding activity by EMSA, CD and polymerase stop assays (PSA) the using DNA sequences capable of forming G4 present in promoters of several proto-oncogenes (including its best described target c-MYC) and RNA sequences capable of forming G4 in translational control regions present in proto-oncogenes mRNAs. In most of them we observed that CNBP promotes the unfolding of G4 and that CNBP preferentially binds to unfolded sequences rather than to sequences folded as G4. These results suggest that CNBP unfolds G4 through binding unpaired guanine-rich sequences thereby displacing the folded-unfolded G4 equilibrium towards the unfolded state. Additionally, they open the possibility that CNBP may participate in the fine-tuning of G4-dependent proto-oncogens expression regulation.