G-Quadruplexes Regulate miRNA Biogenesis in live zebrafish embryos.

STEEMAN, TOMÁS J.; WEINER, ANDREA M. J.; DAVID, ALDANA; CALCATERRA, NORA B.; ARMAS, PABLO

Workshop; Innovative therapeutic targets in non-canonical nucleic acids structures - winter international school; 2021

RNA guanine quadruplexes (G4s) may function as regulators of mRNAs metabolism, processing, and translation [1]. G4s formed within pre-miRNAs (precursors of microRNAs) may compete with the classical stem-loop structure and impair miRNA maturation [2]. Since miRNAs play important roles in the regulation of gene expression during embryonic development, here we use zebrafish embryos as a model to study in vivo G4 biological consequences on miRNA biogenesis and function. We performed an in silico search of putative G4 sequences (PQSs) in pre-miRNAs reported for zebrafish using the miRBase and Ensembl databases and the Quadparser program with the consensus G3-5N1-7G3-5N1-7G3-5N1-7G3-5N1-7. We identified one miRNA (miR-150) whose pre-miRNA contains an evolutionary conserved PQS that is part of the predicted stem-loop. Through circular dichroism spectroscopy, we showed that this PQS folds in vitro as G4. One of the best described and conserved targets of miR-150 is c-myb, a gene involved in proliferation and differentiation of hematopoietic progenitors with a well-defined knock-down phenotype in zebrafish development [3-4]. We performed in vivo analysis of the function of the G4 structure through the overexpression by microinjection of the in vitro transcribed pre-miR-150 (capable of folding as G4) or 7dG-pre-miR-150 (unable to form G4, synthesized using the nucleotide analog 7-deaza-GTP instead of GTP) [5]. 7dG-pre-miR-150 injected embryos showed higher levels of miR-150 and lower levels of c-myb mRNA than those embryos injected with pre-miR-150. Moreover, larvae microinjected with 7dG-pre-miR-150 showed stronger phenotypes associated with c-myb loss-of-function (reduced eye size and larvae length). Results suggest that the G4 formed in pre-miR-150 may function in vivo as a regulatory structure that may compete with the stem-loop structure necessary for miRNA maturation. Future experiments will try to better characterize the structural relationship between G4 and stem-loop, as well as completing the in vivo analysis by characterization of more c-myb related phenotypes.