Identification and selection of pre-miRNAs containing PQSs. (a) Diagram of the computational search strategy of zebrafish pre-miRNAs sequences containing PQSs. (b) Sequence and stem-loop structure of zebrafish pre-miR-150. The mature miR-150 sequence (pink), the PQS (blue), the synthetic RNA oligonucleotide used in subsequent experiments (green), and the G-tracts (yellow boxes) are detailed.

In vitro evidence of the formation of a G4 structure in the PQS present in pre-miR-150. (a) CD spectroscopy of the folded RNA oligonucleotide containing the pre-miR-150 PQS in different K⁺ and Li⁺ concentrations. (b) TDS spectrum of the RNA oligonucleotide containing the pre-miR-150 PQS (70 °C spectrum–20 °C spectrum) folded in the presence of 1 mM K⁺. (c) ThT fluorescence assay with the RNA oligonucleotide containing the pre-miR-150 PQS folded in the presence of 1 mM K⁺. Two-tailed t-Student, *** indicates p < 0.0001. (d) Imino proton region of the 1D ¹H NMR spectrum of the RNA oligonucleotide containing the pre-miR-150 PQS folded in the presence of 1 mM K⁺.

In vivo analysis of the role of the G4 present in pre-miR-150 during zebrafish embryo development. (a) Diagram of the strategy used to disrupt the formation of the G4 in the pre-miR-150 microinjected in zebrafish embryos. Reverse transcription followed by quantitative PCR (RT-qPCR) of miR-150 (b), myb (c), and miR-133a (d) using RNA samples from 24 hpf staged embryos microinjected with miR-150 or miR-133a precursors synthesized using either GTP (G-pre-miR-150 and G-pre-miR-133a) or 7-Deaza-GTP (7DG-pre-miR-150 and 7DG-pre-miR-133a). Ordinary 1-way ANOVA with Tukey multiple comparisons test, p < 0.005. Micrographs of representative 48 hpf staged larvae (lateral view, head to the left) microinjected with the RNA transcribed from pSP64T+dsRED plasmid containing no cloned pre-miRNA (control, (e)) and 7DG-pre-miR-150 (f), with the measured parameters indicated by blue, dashed lines in (e). Trunk length (g) and eye size (h) measurements of 48 hpf staged larvae microinjected with G-pre-miR-150 or 7DG-pre-miR-150. Ordinary 1-way ANOVA with Tukey multiple comparisons test, p < 0.001. Letters above each condition in the graphs represent statistical groups, where means with no letter in common are significantly different. For graphs shown in (b–d,g,h) p-values are detailed in Supplementary Table S7 and numerical values are detailed in Supplementary Table S8. Data shown represent results of one from three independent experiments.

In vitro effect of PDS on pre-miR-150 G4 stability. (a) CD melting curves of the RNA oligonucleotide containing the pre-miR-150 PQS in presence of 1 mM K⁺ and different PDS concentrations. The Tm for 5 and 10 µM PDS could not be calculated due to the absence of a clear melting transition. CD spectra of the RNA oligonucleotide containing the pre-miR-150 PQS folded in presence of 1 mM K⁺ and incubated with different PDS concentrations at the start ((b), 20 °C) and at the end ((c), 95 °C) of the melting experiments.

In vivo effect of PDS on pre-miR-150 G4 during zebrafish embryo development. RT-qPCR quantification of miR-150 (a) and myb (b) of 24 hpf staged embryos microinjected with G- or 7DG-miR-150 in the absence or in the presence of 5 µM PDS. Ordinary 1-way ANOVA with Tukey multiple comparisons test, p < 0.001. Trunk length (c) and eye size (d) measurements of microinjected 48 hpf staged larvae microinjected with G- or 7DG-miR-150 in the absence or in the presence of 5 µM PDS. Ordinary 1-way ANOVA with Tukey multiple comparisons test, p < 0.001. Letters above each condition in the graphs represent statistical groups, where means with no letter in common are significantly different. For graphs shown in (a–d), p-values are detailed in Supplementary Table S7 and numerical values are detailed in Supplementary Table S8. (e) Micrographs of representative 48 hpf staged larvae (lateral view, head to the left) microinjected with the RNA transcribed from pSP64T+dsRED plasmid containing no cloned pre-miRNA (control), G-pre-miR-150, G-pre-miR-150 + PDS, 7DG-pre-miR-150, and 7DG-pre-miR-150 + PDS. The measured parameters for graphs in Figure 5c,d are indicated by blue, dashed lines in the control. Data shown represent results of one from three independent experiments.