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

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

Praga

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

6th International Meeting on Quadruplex Nucleic Acids.

G-quadruplexes(G4) have been found overrepresented in G-rich nucleotide sequences not only intranscriptional control DNA regions, but also in translational control RNAelements of human proto-oncogenes. The fine-tuning of G4 formation in generegulatory regions has emerged as a novel natural and artificial way toregulate gene expression. Here we report the role of cellular nucleic acidsbinding protein (CNBP) on the formation of G4 relevant for expressionregulation of proto-oncogenes. CNBP is a highly conserved vertebrates? proteinwith nucleic acids chaperone activity that has been implicated intranscriptional control through binding to ssDNA and translational controlthrough binding to RNA (1,2,3). Moreover, this protein has been implicated incraniofacial development and in several human diseases (e.g. myotonic dystrophytype 2, sporadic inclusion body myositis and Treacher Collins syndrome) (1,4).In this work we analyzed CNBP binding and biochemical action on a paralleltetramolecular G4 formed by the DNA sequence TGGGGT using electrophoreticmobility shift assays (EMSA), circular dichroism (CD) spectroscopy,helicase-PAGE assays and 1D 1H-NMR, and we observed that CNBP has anATP-independent G4-unfolding activity. Then we tested CNBP binding and G4-unwindingactivity by EMSA, CD and polymerase stop assays (PSA) using DNA sequencescapable 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 foundin proto-oncogenes mRNAs. In most of them we observed that CNBP promotes theunfolding of G4 and preferentially binds to unfolded sequences rather than tosequences folded as G4. These results may indicate that CNBP unfolds G4 throughbinding unpaired guanine-rich sequences thereby displacing the folded-unfoldedG4 equilibrium towards the unfolded state. Additionally, they suggest that CNBPmay participate in the fine-tuning of G4-dependent proto-oncogenes expressionregulation.