Fig. 1. Identification of synaptic target genes of miR-1908-5p and validation of binding sites with luciferase assays. (a) GO analysis of putative miR-1908-3p target genes. The 481 miR-1908-3p targets predicted by TargetScan were narrowed down to 184 genes by selecting those with both context++ scores less than −0.2 and high brain expression (left panel). GO analysis with the 184 miR-1908-3p targets showed significant terms including 'regulation of cell development' and 'regulation of axonogenesis' in the Biological Process category (right panel). (b) GO analysis of putative miR-1908-5p target genes. The 2,500 miR-1908-5p targets predicted were narrowed down to 225 genes by selecting those with both context++ scores less than −0.5 and high brain expression (left panel). GO analysis with the 225 miR-1908-5p targets revealed 'synapse' as the most significant term in the Cellular Component category (right panel). (c) Pre- and post-synaptic localization of the putative miR-1908-5p target genes (color coded, upper panel). Some of the interacting proteins (e.g. PSD-95) of miR-1908-5p synaptic targets are also shown (gray). We selected six synaptic targets of miR-1908-5p for further analysis (lower panel). (d) Luciferase assays with the 3'UTRs of six synaptic targets of miR-1908-5p. The putative miR-1908-5p binding sites in the 3'UTRs are shown (upper panel). miR-1908-5p decreased the luciferase activities of constructs with DLGAP4, GRIN1, STX1A, CLSTN1, and GRM4 3'UTRs but not with GRASP 3'UTR (lower panel). (n=6 from three independent experiments). (e) Validation of the miR-1908-5p binding sites in the GRM4 3'UTR. Mutations of both binding sites (M1/2), but not each binding site alone (M1 or M2), blocked the inhibitory effect of miR-1908-5p on the expression of GRM4 3'UTR. (n=6 from three independent experiments). (f) Conservation of the first (770~776) and second (803~809) miR-1908-5p binding sites in the GRM4 3'UTR. The species with miR-1908-5p binding seed sequences are red-colored. (g) Conservation of the miR-1908-5p sequence. The species with miR-1908-5p seed sequences are red-colored. All data are presented as mean±SEM. Statistical analyses are in Additional file 1: Table S1.
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