GENETIC POLYMORPHISMS ON G-QUADRUPLEXES AS A CAUSE OF ONCOGENES TRANSCRIPTIONAL AND TRANSLATIONAL EXPRESSION VARIATIONS.

BEZZI, GEROGINA; NORA B. CALCATERRA; PIGA, ERNESTO; BINOLFI, ANDRES; LORENZATTI, AGUSTÍN; PABLO ARMAS

Congreso; LV Annual SAIB Meeting and XIV PABMB conference; 2019

G-quadruplexes (G4) are nucleic acid secondary structures that can be transiently folded within proximal promoter regions (PPRs) in G-rich single-stranded DNA regions exposed during transcription and in G-rich RNA sequences within 5' untranslated regions (5? UTRs) or other mRNA regions involved in translational control. G4s have been proposed as novel transcriptional and translational regulatory elements originally and mainly described in oncogenes. On the other hand, genomic scale association studies by massive DNA sequencing revealed that single nucleotide polymorphisms (SPNs) associated with human diseases are present mainly near transcription start sites, within PPRs and 5' UTRs. The goal of this work was to identify SNPs overlapped with putative G4 forming sequences (PG4) described as transcriptional or translational regulators (located within PPRs or 5' UTRs, respectively) of oncogenes, that may affect G4 folding, hereafter called SNP-PG4. First we performed a bioinformatic analysis using Ensembl database to identify the SNPs (reported in COSMIC, ClinVar, dbSNP and HGMD genetic variation databases) overlapped with the PG4s (and their +/- 5 bp flanking sequences), described as transcriptional regulators for 10 oncogenes and as translational regulators for 15 oncogenes. For each reference sequence we generated a collection of variable sequences containing each polymorphism and a mutant sequence with no PG4 (unable to form G4). Then we used several DNA and RNA G4 folding predictors in order to identify those SNP-PG4 that may affect G4 folding or stability. Based on the results of this analysis, from 88 DNA and 256 RNA sequences corresponding to the SNP-PG4 of the analyzed oncogenes, we chose 41 and 15, respectively, for further analysis. The selected sequences correspond to the c-MYC, BCL2, cKIT, RET and VEGF oncogenes for G4s in PPRs and to the CCND3, NRAS, HSAFY, ESR1, FGF2, ZIC1 and TRF2 oncogenes for G4s in 5? UTRs. Spectroscopic analyses by Circular Dichroism (CD) demonstrated that some SNPs cause quantitative or qualitative spectral changes. Moreover, qPCR stop assays and CD melting assays indicate that the same SNPs induce G4 stability changes. In agreement, 1D 1H NMR spectroscopy confirmed that SNPs induce quantitative and qualitative changes for the SNP-PG4s identified for c-MYC and NRAS. Finally, SNP-PG4s that produced significant structural variations in vitro were cloned into pGL3 promoter vector (for PG4s controlling transcription) or into psiCHECK-2 vector (for RNA PG4 controlling translation) and were transfected into HEK293 cells, revealing that SNPs altered luciferase reporter activity. Results gathered in this work suggest that SNP-PG4s that alter G4 folding may be the cause of differential expression of oncogenes leading to tumor predisposition, establishment, progression or metastasis and should be considered as a novel molecular etiology mechanism for the predisposition or establishment of diseases.