Consequences of disease-related genetic variations in guanine quadruplexes on post-transcriptional gene expression

BAYON, C; GISMONDI, M.; MARGARIT, E.; ARMAS, P.

Rosario

Congreso; LIX Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2023

SAIB

Consequences of disease-related genetic variations in guanine quadruplexes onpost-transcriptional gene expressionBayon, Candela1; Gismondi, Mauro2; Margarit, Ezequiel2; Armas, Pablo1.1Instituto de Biología Molecular y Celular de Rosario (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas(CONICET), Universidad Nacional de Rosario (UNR), Rosario S200EZP, Santa Fe, Argentina. 2Centro de EstudiosFotosintéticos y Bioquímicos (CEFOBI), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Rosario,Santa Fe, Argentina.bayon@ibr-conicet.gov.arGuanine quadruplexes (G4s) are non-canonical nucleic acid structures playing pivotal roles in modulating gene expressionmechanisms ranging from transcription to translation, as well as other DNA- and RNA-associated processes. G4s are formedwithin G-rich single-stranded sequences characterized by a pattern of four tracts of 3 or more Gs separated by loops of 1 to 12nucleotides (G3+N1-12G3+N1-12G3+N1-12G3+). Each G within a G-tract interacts with Gs in the other tracts to form aG-tetrad through Hoogsteen bonds, which can subsequently stack adopting the G4 structure. The stability of a G4 is closelyrelated to the number of stacked G-tetrads and the length and composition of the loops as well as the adjacent sequences. PutativeG4-forming sequences (PG4s) are overrepresented within the human genome's in gene regulatory regions, particularly thoseassociated with proto-oncogene transcription and translation control. G4s folding dynamics can be affected by short geneticvariations within these PG4s. Moreover, disease related variants in noncoding regions can alter gene expression, prompting thehypothesis that these variants affect regulatory motifs, possibly G4s. To identify genetic variants in PG4s impacting G4 formationand stability in translation control, we conducted a genomic search on 5' UTRs and CDSs using the ENSEMBL database. Thedownloaded metadata were processed using R and Perl programming languages, which allowed us to identify flanking sequencesto short genetic variants containing RNA PG4s. We then classified the variants into two groups. Group "A" corresponds tovariants that present PG4 only in the sequence with the reference allele and not in the sequence with the alternative allele, thusincludes variants that may cause the disruption of G4s formed in the reference sequences. On the other hand, group "B"corresponds to variants that present PG4 only in the alternative allele-sequence and not in the reference allele-sequence, thusincludes variants favoring the formation of G4s not formed in reference sequences. PG4s associated with variants from groups“A” and “B” were assigned G4 formation scores using the G4RNAScreener predictor, which were used together withbibliographic data for selecting five candidates from group "A" to characterize in vitro the effect of the identified variants on theformation and stability of G4s. By means of Circular Dichroism spectroscopy, we observed that the PG4s found in the referencesequences can form G4 structures and present quantitative differences in the spectroscopic signals and structural stabilitiescompared to the alternative sequences. Our work suggests that variants affecting G4 folding may impact differential geneexpression at the translational level, potentially contributing to human disease predisposition or onset. The effects of thesevariants on G4-mediated translation will be assessed through reporter gene expression assays in cultured cells.Keywords: RNA, G-quadruplex, Short Genetic Variations, Translation, DiseaseMethods: Bioinformatics sequence analysis, Circular Dichroism (CD) Spectroscopy, CD melting assays.