Fig 1: Several initiation factors are directly targeted by miR-322/miR-503. (A) Putative miR-322/miR-503 recognition sites on the 3'-UTR of eIF4E, eIF4G1, eIF4B, eIF2B5, and eIF3M, as predicted by TargetScan. (B) miR-322/miR-503 inhibited luciferase activity of a firefly luciferase reporter fused with 3'-UTRs from eIF4G1, eIF4B, eIF2B5, and eIF3M, but not eIF4E. Mutation of the putative recognition sites abolished the inhibition. (C) Confirmation of the down-regulation of eIF4E, eIF4G1, eIF4B, eIF2B5, and eIF3M in additional transgenic muscles by RT-PCR (n = 5 vs. 5). (D) Expression of eIF4E, phospho-eIF4E, eIF4G1, and eIF4B proteins was down-regulated in transgenic muscles. The expression of phospho-eIF2a was unchanged. (E) Quantification of protein expression levels of eIFs (n = 4 vs. 4). (F) New protein synthesis was inhibited in transgenic muscles as determined by SUnSET (surface sensing of translation). Mice were labelled with 0.04 µmol/g puromycin for 30 min. (G) Quantification of levels of puromycin-tagged peptides (n = 4 vs. 4). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.