Proposed model of miR-486 regulation and function in muscle cells. miR-486 transcription is activated by MyoD and MRTF-A. miR-486 directly represses the translation of PTEN and Foxo1a, two crucial negative regulators of PI3K/Akt signaling, resulting in the phosphorylation of Akt and the activation of the pathway. Activated Akt results in the phosphorylation of GSK3β and the inhibition of Foxo1a activity.

Activation of PI3K/Akt signaling by MRTF-A and miR-486. (A) The endogenous protein levels of PTEN and phosphorylated Akt were detected by Western blot from CMCs transduced with 10 MOI of control or MRTF-A–expressing adenovirus. The intensity of the band for PTEN, normalized to GAPDH and relative to control infection, is depicted below the image. (B) The endogenous protein levels of PTEN, phosphorylated GSK3β, phosphorylated Akt, total Akt, and GAPDH were detected by Western blot from CMCs transduced with 1 MOI of MRTF-A and/or 10 MOI of STARS-expressing adenovirus. The intensity of the band for PTEN, normalized to GAPDH and relative to control infection, is depicted below the image. (C) siRNA-mediated knockdown of miR-486 in CMCs demonstrated by real-time RT-PCR and normalized to that of U6 expression. si-miR-486 abolishes the induction of miR-486 expression in response to MRTF-A. (D) The endogenous protein levels of PTEN, Foxo1a, phospho-Akt, total Akt, and GAPDH were detected by Western blot in si-miR-486 treated CMCs. (Upper) Transduction of CMCs with 10 MOI of control or MRTF-A–expressing adenovirus. si-miR-486 or control transfection is indicated at the top by “+” or “−,” respectively. Intensity of the band for PTEN or Foxo1a, normalized to GAPDH and relative to the control lane, is shown below the image.

miR-486 and host gene, sAnk1, are induced by MRTF-A. (A) Cardiac myocytes (CMCs) were infected with adenovirus directing expression of β-gal (−) or MRTF-A (+). miR-486 was detected by Northern blot. (B) The response of miR-486 and sAnk1 to increasing multiplicity of infection (MOI) of adenovirus-mediated MRTF-A expression in CMCs was examined by real-time RT–PCR. Expression levels are relative to that in β-gal-infected control CMCs. (C) miR-486 is encoded by the final intron of the Ank1 gene (Top), which is regulated by an alternative promoter containing two E-boxes. The first intron contains two CArG boxes. (Middle) The genomic organization of the Ank1 gene and miR-486 (red box). (Bottom) The genomic organization of the muscle-enriched sAnk1 isoform, including the putative regulatory elements.

Muscle-specific expression of miR-486 and sAnk1. (A) Northern blot of miR-486 in adult mouse tissues. RT–PCR demonstrates the adult expression of the miR-486 host gene sAnk1. Controls for RNA input in the Northern blot and RT–PCR are U6 and GAPDH, respectively. (B) In situ hybridization demonstrates embryonic and adult expression of the sAnk1 gene. (i) sAnk1 is observed specifically in the developing somites and limb muscles of a whole-mount E11.5 embryo; head staining is nonspecific. Radioactive-section in situ hybridization demonstrates sAnk1 expression at E12.5 (ii) and in the embryonic and adult heart (iii and iv). (ii) sAnk1 is robustly expressed in the somites, tongue, facial muscles, and diaphragm. Expression within the heart is localized to the atria, AV node, and bundle branches at E17.5 (iii). sAnk1 expression is observed throughout all cardiac chambers in the adult heart (iv). (Scale bar: 1 mm.) at, atrium; avn, atrioventricular node; bb, bundle branches; di, diaphragm; fm, facial muscles; lm, limb muscle; s, somites; tg, tongue; v, ventricle.

miR-486 is predicted to target PTEN and Foxo1a. (A) Normalized expression level of miR-486 in the ventricles detected by real-time RT-PCR at various postnatal ages and represented relative to postnatal day 0 (PO) in a line graph. Normalized levels of PTEN protein are plotted as a percentage of the level at P0. (B) The level of endogenous PTEN protein from ventricular lysates of indicated postnatal ages was detected by Western blot. PTEN band intensity, normalized to GAPDH and relative to P0, is shown below image of Western blot. (C) miR-486 targets the 3′ UTRs of PTEN and Foxo1a mRNAs. The 3′ UTR of the PTEN and Foxo1a mRNAs was linked to the pMIR-REPORT luciferase plasmid (Ambion) and examined in COS cells transiently transfected with CMV6-miR-486 or CMV6-β-gal as a negative control. “mut” refers to mutation of the predicted miR-486 seed region. *P value < 0.05. Error bars represent the SD. (D) Adenoviral-mediated overexpression of miR-486 in CMCs demonstrated by Northern blot. Arrowhead marks the precursor miRNA. U6 was detected as the control for RNA input. (E) The endogenous protein levels of PTEN, phospho-Akt, and total Akt were detected by Western blot in the control or miR-486–transduced CMCs. GAPDH was detected as the control for protein loading.

Identification of miR-486/sAnk1 regulatory sequences in vitro. (A) Luciferase assay demonstrating the response of 1080 bp of upstream DNA (WT) or of the DNA fragment containing a mutation of the E-boxes to increasing amounts of MRTF-A and MyoD. A total of 50, 100, and 200 ng of expression plasmids were cotransfected with the luciferase reporter construct in COS cells. Error bars represent SD. (B) Response of the 649 bp of sAnk1 intron 39a to MRTF-A examined by luciferase reporter assay in COS cells. A total of 50, 100, and 200 ng of expression plasmid were transfected with full-length intron–luciferase (WT), a luciferase reporter construct with a mutation in the proximal CArG1, a mutation in the distal CArG2, or a truncation that consists of the 5′ most 300 bp. Error bars represent the SD. (C) Response of the empty luciferase vector or the 1,080-bp upstream luciferase or various intron–luciferase reporters to adenovirus-mediated MRTF-A expression in CMCs. Error bars represent SD. (D) Gel electromobility shift assay demonstrating the binding of flag epitope-tagged SRF overexpressing COS cell lysate to a radiolabeled oligonucleotide probe consisting of the sequences of the CArG1 or CArG2 of sAnk1 intron 39a. Flag antibody results in a supershift, and wild-type unlabeled competitor abolishes the binding of CArG probe. Mutant probe does not bind SRF, nor does mutant unlabeled competitor abolish WT probe binding to SRF. m, mutant CArG.

Identification of miR-486/sAnk1 regulatory sequences in vivo. (A) Embryonic and adult transgenic mice harboring a lacZ transgene controlled by 1080 bp upstream of the sAnk1 gene exhibit skeletal-muscle–specific β-gal activity. Reporter expression is observed (i) in somites and facial muscles at E11.5 and (ii) in the entire developing musculature at E13.5. (iii) Dorsal view of a cleared E13.5 embryo demonstrating internal muscle staining and absence of smooth muscle or cardiac staining. (iv–vi) lacZ expression detected in whole-mount (iv) GP, (v) TA, and (vi) EDL muscles of adult transgenic mice. (Scale bar: 1 mm.) (B) Adult transgenic mouse harboring a lacZ transgene controlled by the 5′-most 300 bp of intron 39a of the sAnk1 gene. Expression of lacZ is observed in the (i) atria and AV canal of the heart and in the (ii–v) GP, TA, Sol, and EDL of adult mice. (i) Arrows mark AV canal. (Scale bar: 1 mm.) EDL, extensor digitorum longus; GP, gastrocnemieus plantaris; Sol, soleus; TA, tibialis anterior. (C) Summary of the transgenic constructs used to examine the regulation of sAnk1/miR-486. Number of transgenic embryos displaying muscle-specific expression per total number of transgenic embryos is shown in right columns. Embryonic and adult expression was assessed in F₀ transgenics and stable transgenic lines. The 1,080-bp upstream reporter containing the two E-boxes directed embryonic and adult skeletal muscle expression, but was insufficient for cardiac expression. The full-length intron reporter, containing two CArG boxes, was insufficient to direct expression in transgenic mice; however, a truncated intron consisting of the 5′ most 300 bp and a single CArG box directed adult skeletal muscle and heart expression. Mutation of the single CArG box within the truncated intron abolished lacZ expression in the skeletal muscle and heart.