Figure 1. X‐linked miR‐506 family miRNAs promote FMRP expression in mouse spermatogonia.

(A) Schematic illustration of the strategy used for generation of the whole miR‐506 miRNA cluster KO mice using CRISPR‐Cas9. The red lightning bolt represents the gRNAs used, and its right and left orientations indicate the gRNAs targeting the reverse and forward strands of the genomic DNA, respectively. Green and black arrows indicate precursor miRNAs and miRNA clusters, respectively. The whole cluster KO was achieved through sequential deletion of individual clusters, and the number inside the parenthesis shows the order of CRISPR‐Cas9‐based deletion. (B) Heatmap showing the abundance of the miR‐506 family miRNAs in spermatogonia and Sertoli cells based on sRNA‐seq. Two replicates were done for each cell type. (C) TaqMan probe‐based qPCR analyses of the miR‐506 family miRNAs in WT and KO adult testes. miR‐16‐5p was used as a loading control. Expression levels were normalized to U6. Data are presented as means ± SEM, n = 3. (D) Heatmap of small RNA‐seq data showing expression levels of the miR‐506 family miRNAs in WT and KO adult testes. Testes of biological triplicates were used for small RNA‐seq analyses. (E) qPCR analyses of Fmr1 and Slitrk2 mRNA levels in WT and KO adult testes. Expression levels were normalized to Gapdh. Data are presented as means ± SEM, n = 3. (F) A representative image of Western blots of FMRP and GAPDH in WT and KO testes. Upper panel, blots of FMRP and GAPDH proteins; lower panel, Ponceau S staining of the membrane. (G) Quantitative analysis of FMRP levels based on Western blot results. The density of the bands was analyzed using ImageJ, and FMRP levels were normalized to GAPDH. Data are presented as means ± SEM, n = 3. *, **, and *** indicate P < 0.05, 0.01, and 0.001, respectively.