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

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Genomic DNA as well as RNA molecules may transiently fold as G-quadruplexes (G4s), non-canonical structures of nucleic acids associated with gene expression control and genome integrity. Although G4s formation and function was demonstrated in vitro and in cultured cells, the in vivo biological relevance of these structures is still elusive. Here, we used zebrafish embryonic development as an in vivo model to assess the role of G4s on the translation of developmentally regulated genes. The objective of this study was to identify conserved putative G4 forming sequences (PG4s) among H. sapiens, M. musculus, and D. rerio in genes 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 developmentally regulated genes containing conserved PG4s within their 5´UTRs (zeb1a), CDSs (megf8 and col2a1) and 3´UTRs (smad3a) and assayed by circular dichroism spectroscopy that they are able to fold as G4s in vitro. Then, we cloned human and zebrafish PG4s of zeb1a and megf8a upstream and within, respectively, Renilla luciferase reporter CDS, and transfected the constructs in HEK293 cell line. Results reveal that all tested G4s alter Renilla luciferase activity by affecting translation levels. Finally, for the case of megf8, the in vivo role of the G4 was analyzed by microinjection experiments in zebrafish embryos of in vitro generated mRNAs containing the PG4s fused to GFP reporter coding sequence and specific disruption by co-microinjection of antisense oligonucleotides (ASOs) complementary to the PG4s thus disrupting G4 formation. This strategy led to translational decrease of the analyzed fluorescence expression, indicating that in this biologically relevant environment, the G4 of megf8 may act as a translation activator. Overall, this study indicates that G4s function regulating translation in vivo and may act as conserved fine-tuning elements of gene expression during embryonic development.