G-Quadruplexes regulate miRNA-150 biogenesis.

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

Congreso; Congreso conjunto SAIB-SAMIGE 2021; 2021

Guanine quadruplexes (G4s), are non-canonical nucleic acids secondary structures formed by the stacking of at least two guanine tetrads bonded by Hoogsteen hydrogen bonds. Intramolecular G4s are dynamic structures transiently formed in G-rich ssDNA or RNA. RNA G4s have been reported as regulators of mRNAs translation and RNAs metabolism and processing, participating in processes such as splicing, polyadenylation of pre-mRNA and maturation and function of microRNAs (miRNAs). Although the formation and function of G4s have been demonstrated in vitro and in cellulo, their biological relevance in vivo has been little explored. It has been reported that G4s formed by pre-miRNAs (precursors of miRNAs that will be processed by the Dicer ribonuclease to produce miRNAs) may compete with the classical stem-loop structure and reduce Dicer capability to generate the mature miRNAs. Since miRNAs play important roles in the regulation of gene expression during embryonic development, here we use zebrafish embryonic development as a model to study 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 a conserved PQS that is part of the predicted stem-loop and it is partially complementary to the mature miRNA. Through circular dichroism spectroscopy, we showed that this PQS folds in vitro as G4, which could interfere in the processing of the miR-150 by acting as a regulatory element. 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. We performed in vivo analysis of the function of the G4 structure as regulator of the biogenesis of miR-150 during zebrafish embryonic development through the overexpression by microinjection of the in vitro transcribed pre-miR-150 (capable of folding as G4) or the same RNA in a form unable to fold as G4 (7dG-pre-miR-150, synthesized using the nucleotide analog 7-deaza-GTP instead of GTP). 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. 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 Dicer processing. Near future experiments will try to better characterize the structural relation between G4 and stem-loop, as well as completing the in vivo analysis by characterization of the phenotypes related to thrombocyte-erythrocyte lineage determination in zebrafish larvae overexpressing miR-150 (and reduced in c-myb levels) as a consequence of the microinjection of pre-miR-150 or 7dG-pre-miR-150.