Guanine Quadruplexes Involved in Post-Transcriptional Regulation of Gene Expression Relevant to Embryonic Development

BEZZI, G; GISMONDI, M.; MARGARIT, E.; ARMAS, P.

Rosario

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

SAIB

Guanine Quadruplexes Involved in Post-Transcriptional Regulation of Gene ExpressionRelevant to Embryonic DevelopmentBezzi, Georgina1; 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, Santa Fe, Argentina. 2Centro de Estudios Fotosintéticos yBioquímicos (CEFOBI), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Rosario, Santa Fe, Argentina.bezzi@ibr-conicet.gov.arGenomic DNA as well as RNA molecules may transiently fold as G-quadruplexes (G4s), non-canonical structures of nucleicacids associated with gene expression control and genome integrity. Although G4s formation and function was demonstrated invitro and in cultured cells, the in vivo biological relevance of these structures is still elusive. Here, we used zebrafish embryonicdevelopment as an in vivo model to assess the role of G4s on the translation of developmentally regulated genes. The objective ofthis study was to identify conserved putative G4 forming sequences (PG4s) among H. sapiens, M. musculus, and D. rerio ingenes related to embryonic development and located in regions probably implicated in the translational regulation of these genes.Firstly, we conducted a bioinformatic analysis using the Ensembl database to identify ~1100 genes related to the Gene Ontology(GO) term 'embryo development' for each species. Subsequently, PG4s were identified within the 5' untranslated region (5'UTR),coding sequence (CDS), and 3' untranslated region (3'UTR) using an algorithm to search PG4s. We found four developmentallyregulated genes containing conserved PG4s within their 5´UTRs (zeb1a), CDSs (megf8 and col2a1) and 3´UTRs (smad3a) andassayed by circular dichroism spectroscopy that they are able to fold as G4s in vitro. Then, we cloned human and zebrafish PG4sof zeb1a and megf8a upstream and within, respectively, Renilla luciferase reporter CDS, and transfected the constructs inHEK293 cell line. Results reveal that all tested G4s alter Renilla luciferase activity by affecting translation levels. Finally, for thecase of megf8, the in vivo role of the G4 was analyzed by microinjection experiments in zebrafish embryos of in vitro generatedmRNAs containing the PG4s fused to GFP reporter coding sequence and specific disruption by co-microinjection of antisenseoligonucleotides (ASOs) complementary to the PG4s thus disrupting G4 formation. This strategy led to translational decrease ofthe analyzed fluorescence expression, indicating that in this biologically relevant environment, the G4 of megf8 may act as atranslation activator. Overall, this study indicates that G4s function regulating translation in vivo and may act as conservedfine-tuning elements of gene expression during embryonic development.Keywords: G-quadruplexes, Embryonic Development, Zebrafish, Translation controlMethods: Circular Dichroism (CD) spectroscopy, CD melting assays, Cell culture, Cell Transfection, Embryo Microinjection.