Fig 2: Transcriptomic profiling of miR-1 KO skeletal muscle and identification of bona fide miR-1 target genes.(A) Cumulative density function (CDF) of AGO2 eCLIP-seq-defined miR-1 targets (green) Log2FC compared to non-miR-1 targets (blue). Zoomed in figure emphasizes rightward shift of miR-1 targets, indicated by arrow. RNA-seq data for integration with AGO2 eCLIP-seq are from gastrocnemius muscles (n = 4 female WT, n = 4 female miR-1 KO mice). (B) Pathway enrichment analysis of significantly up-regulated genes (false discovery rate, FDR <0.05, Log2 fold change, FC > 0) in miR-1 KO/WT that are AGO2 eCLIP-seq-defined miR-1 target genes. (C–D) miR-1 binding peaks in (C) polypyrimidine tract-binding protein 1 (PTBP1) and (D) pyruvate kinase M1/2 (PKM) mRNA outlined with red dotted line, miR-1 alignment on mouse target sequence shown below. (E) Representative Western blot of PTBP1 and corresponding total protein levels in WT and miR-1 KO. (F) Quantification of PTBP1 protein levels after densitometric analysis of the levels of each sample normalized to corresponding total protein levels, expressed as fold-change (FC). (G) Representative Western blot of PKM1 and PKM2, (H–I) Quantification of (H) PKM1 and (I) PKM2 protein levels. (J–K) miR-1 binding peaks in (J) lactate dehydrogenase A (LDHA) and (K) solute carrier family 16 member 3 (SLC16A3) mRNA. (L) Western blot of LDH, (M) quantification of LDH protein levels. (N) Western blot of monocarboxylate transporter 4 (MCT4), (O) quantification of MCT4 protein levels. For Western blotting experiments, gastrocnemius muscle lysates from n = 4–8 WT and n = 4–8 miR-1 KO females were used, and differences between WT and miR-1 KO were tested using an independent t-test (2-tailed). ∗p < 0.05, ∗∗∗p < 0.01, ∗∗∗∗p < 0.0001, ns: not significant.