Fig. 5

bmal1b is the target of miR-219-5p.A The binding site (red) of miR-219-5p in the zebrafish bmal1b 3?UTR. Those of BMAL1/Bmal1 of humans, mice, and rats have one mismatch at the second position. B Schematic diagram of the RNA-binding protein immunoprecipitation (RIP) by Ago2-His, created in BioRender (https://BioRender.com/b76b549). Electrophoretic (C) and qRT-PCR (D) analyses of RIP show that both miR-219-5p and bmal1b are present in the RISC pulled down by Ago2. Data are mean ± SD. n = 3. Unpaired two-tailed Student?s t-test was performed, *P < 0.05, **P < 0.01, ***P < 0.001 vs. Input. E Schematic diagram of the miRNA pull-down by 3?Biotin-tagged miR-219-5p, created in BioRender (https://BioRender.com/b76b549). Electrophoretic (F) and qRT-PCR (G) analyses of miRNA pull-down show that miR-219-5p binds to bmal1b. Data are mean ± SD. n = 3. Unpaired two-tailed Student?s t-test was performed, *P < 0.05 vs. Input. H, I Luciferase reporter assays show that miR-219-5p inhibits bmal1b. Construction of the plasmids of the bmal1b 3?UTR fused with luciferase (WT) and the mutated bmal1b 3?UTR with the deleted miR-219-5p binding site, fused with luciferase (MT) (H). Luciferase assays with NC mimic and miR-219-5p mimic show that miR-219-5p inhibits bmal1b (I). Data are mean ± SD. n = 3 × 3. Unpaired two-tailed Student?s t-test was performed, **P < 0.01 vs. NC mimic. J?L In Vivo assays confirm miR-219-5p inhibits bmal1b. Construction of plasmids of pCS2-EGFP-bmal1b-3?UTR and pCS2-mCherry-MCS control plasmids (J). Images of representative embryos injected with CS2-EGFP-bmal1b-3?UTR or pCS2-mCherry-MCS control plasmids, and miR-219-5p mimic or NC mimic (K). Quantification and statistical analysis of the ratio of GFP and mCherry with ImageJ (L). The scale bar is 100 ?m. Data are mean ± SD. n = 3 × 5. Unpaired two-tailed Student?s t-test was performed, ***P < 0.001 vs NC mimic. See also Fig. S6.