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Fig. 3. miR-133 and miR-1/206 regulate the expression of BAF60a and BAF60b variants. (A) Alignment of putative miR-133, or miR-206 and miR-1 target sites in the 3′UTRs of chick (Gallus gallus, Gga), human (Homo sapiens, Hsa) and mouse (Mus musculus, Mmu) BAF60a and BAF60b genes. Nucleotides complementary to the respective miRNA are in red. The seed sequence of miR-133 is complementary to the predicted target site in the BAF60a 3′UTR and this is conserved in all three species. There is little variation outside the seed sequence. The seed sequences of miR-1 and miR-206, which are identical, are complementary to the predicted target site in the chicken BAF60b 3′UTR. In human and mouse BAF60b 3′UTR, fewer nucleotides are complementary to the miR-1 or miR-206 seed sequence, suggesting a non-canonical binding site where nucleotides outside the seed compensate. The number and position of complementary nucleotides outside the seed sequence vary between species and between miR-1 and miR-206, as these miRNAs differ outside the seed. In human/mouse, BAF60a and BAF60b are known as SMARCD1/Smarcd1 and SMARCD2/Smarcd2, respectively. (B) In situ hybridization using LNA probes shows myotome-specific expression of miR-133, miR-206 and miR-1 in HH20 embryos. my, myotome; dm, dermomyotome. Scale bar: 50 μm. (C) Luciferase sensors containing 3′UTR sequences of chick BAF60a or BAF60b were transfected into DF1 cells. Co-transfection of miR-133, miR-1 or miR-206 led to downregulation of luciferase expression compared with controls. Point mutations in the putative target site rendered the sensors non-responsive. Error bars indicate s.d.; *P<0.05 (t-test). (D) Endogenous BAF60a and BAF60b proteins are regulated by miR-133 or miR-1/206 in mouse NIH3T3 cells. Transfection with miR-133 led to reduced BAF60a protein levels; co-transfection of miR-133 with antimiR-133 restored BAF60a protein levels to that of mock transfected controls. Transfection with miR-1 or miR-206 or both led to reduced BAF60b protein levels; co-transfection of miRNAs with the relevant antimiR restored BAF60b protein levels to that of mock transfected controls. Image published in: Goljanek-Whysall K et al. (2014) © 2014. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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