G-quadruplexes in miRNAs biogenesis and function

DAVID, A.P.; ARMAS, P.

Bernal, Buenos Aires, Argentina

Congreso; II Argentine Meeting on Biology of Non-Coding RNAs; 2018

II Argentine Meeting on Biology of Non-Coding RNAs

Guanine quadruplexes (G-quadruplexes or G4), are non-canonical nucleic acids secondary structures formed by the stacking of at least two guanine tetrads bonded by Hoogsteen hydrogen bonds. Intramolecular G4 are dynamic structures transiently formed in G-rich ssDNA or RNA. DNA G4 have been involved in gene expression regulation and genomic instability. RNA G4 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 DNA and RNA G4 have been demonstrated in vitro and in cellulo, their biological relevance in vivo has been little explored. It has been reported that G4 present in the sequence of pre-miRNA (precursors of miRNA that will be processed by the Dicer ribonuclease to produce miRNAs) may compete with the classical stem-loop structure of the pre-miRNA and reduce Dicer capability to generate the mature miRNA. Since miRNAs play important roles in the regulation of gene expression during embryonic development, we will use zebrafish embryonic development as a model biological process to study G4 biological consequences on miRNA biogenesis and function. First we performed an in silico search of putative G-quadruplex sequences (PQS) 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. Using this strategy, we identified one miRNA (miR-150) which pre-miRNA contains a conserved PQS that is part of the predicted stem-loop and it is partially complementary to the mature miRNA. miR-150 was reported to have the conserved target c-myb, a gene involved in proliferation and differentiation of hematopoietic progenitors and with well-defined knock-down phenotype in zebrafish. Then, through circular dichroism, 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. Finally, we present strategies for the in vivo analysis of the function of G4 as regulator of the biogenesis of miR-150 during zebrafish embryonic development based in the overexpression by microinjection of the pri-miR-150 in the wild-type form (capable of folding as G4) or in a form unable to fold as G4 (synthesized using the nucleotide analog 7-deaza-GTP instead of GTP). In microinjected embryos we will evaluate c-myb mRNA and protein levels, as well as phenotypic traits characteristic of c-myb knock-down, like shortened trunk, reduction of the eyes size and slow heart rate.