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. 2021 Oct 26;12(6):2174–2186. doi: 10.1002/jcsm.12827

Figure 4.

Figure 4

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‐eIF2α 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.