Axonal precursor miRNAs hitchhike on endosomes and locally regulate the development of neural circuits (2020)
Figure 1

Figure 1.

Molecular beacons are new, specific tools to detect endogenous pre-miRNAs in cells

A. Schematic representation of the experimental protocol. Abbreviations: LCM, laser capture microdissection.

B, C. RT-PCR performed on RNA extracted from axons collected by LCM (Ax) or from eyes. (B) β-Actin mRNA is present both in eyes and axons, while MAP2 and H4 are present only in the eye sample, suggesting the absence of contamination from cell bodies or other cells in LCM axonal samples (Bellon et al, 2017). Abbreviations: Ax, axonal sample; Eye, stage 37/38 eye; LCM, laser capture microdissection; -, PCR no template control; NT, RT no template control.

D. Schematic of molecular beacon (MB), pre-miR-181a-1 and their hybridization complex. Abbreviations: MB, molecular beacon.

E. Schematic of thermal denaturation profile of the MB. Abbreviations: MB, molecular beacon.

F. Thermal denaturation profiles of MB, in the absence (solid line) and presence (dashed line) of increasing target concentration. Each melting curve represents the average of three separate replicates. Yellow boxes indicate the range of working temperatures for ex vivo trafficking experiments. Abbreviations: MB, molecular beacon; Tm, MB melting temperature.

G. Schematic representation of the experimental protocol. Concentrations used: 5µM MB; 250µM co-MO; 250µM pri-miR-MO. Abbreviations: MB, molecular beacon; co-MO, control morpholino; pri-miR-MO, morpholino blocking pre-miR-181a-1 processing by targeting the Drosha cleavage site.

H. Quantification of the expression levels of pre-mir-181a-1 using the 2-ΔCt method and U6 as normalizer from small total RNA fraction (<~150 nt). Each data point corresponds to one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant.

I. Total number of MB puncta normalized to axon length (µm). Each data point corresponds to one axon. Total number of puncta and axons analyzed: 928 puncta, 61 axons (WT), 226 puncta, 15 axons (co-MO), 208 puncta, 35 axons (MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino; pri-miR-MO, morpholino blocking pre-miR-181a-1 processing by targeting the Drosha cleavage site.

J. Representative axons. MB puncta are indicated (white arrows). Dashed white lines delineate axons. Abbreviations: MB, molecular beacon. Scale bars: 5µm.

K. Schematic representation of the experimental protocol. Concentrations used: 5µM MB; 200-250ng/µL cy3-pre-miR-181a-1. Abbreviations: MB, molecular beacon.

L. Schematic representation of cy5-labeled pre-miR-181a-1.

M. Frequency (in percentage) of puncta colocalization between MB and cy5-pre-miR-181a-1 (#MB+/pre-miR+). Each data point corresponds to one axon. Total number of puncta and axons analyzed: 354 puncta, 32 axons (MB), 337 puncta, 32 axons (cy5-pre-miR-181a-1). n=5 independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon.

N. Representative image of RGC axons. White, red and blue arrows indicate, respectively, co-localized, single MB, and single pre-miR-181a-1 puncta. Scale bars: 5µm.

Data information: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Data were normally distributed (Shapiro-Wilk test), one-way ANOVA followed by Tukey's multiple comparison post-hoc test (H). Data were not normally distributed (Shapiro-Wilk test), Kruskal Wallis followed by Dunn's multiple comparison post-hoc test (I).