Uncoupling of Expression of an Intronic MicroRNA and Its Myosin Host Gene by Exon Skipping

Abstract

The ancient MYH7b gene, expressed in striated muscle and brain, encodes a sarcomeric myosin and the intronic microRNA miR-499. We find that skipping of an exon introduces a premature termination codon in the transcript that downregulates MYH7b protein production without affecting microRNA expression. Among other genes, endogenous miR-499 targets the 3 untranslated region of the transcription factor Sox6, which in turn acts as a repressor of MYH7b transcriptional activity. Thus, concerted transcription and alternative splicing uncouple the level of expression of MYH7b and miR-499 when their coexpression is not required.Skeletal and cardiac muscle exhibit a broad range of contractile characteristics and adaptive responses that are controlled, at least in part, by sarcomeric myosins, a multigene family of actin-based motors that convert chemical energy released from ATP hydrolysis into mechanical force. In addition to the eight well-characterized myosin heavy chain (MYH) isoforms expressed in heart and skeletal muscle, three more ancient myosin genes, MYH7b (MYH14), MYH15, and MYH16, have been identified (13). In humans, the MYH16 gene is a pseudogene by virtue of a frameshift mutation and consequent premature termination codon (PTC). Remarkably, the gene is not mutated in other nonhuman primates and appears to regulate the fiber size of the masticatory muscles (35). Phylogenetic analysis suggests that MYH7b is most closely related to ␣-and ␤-cardiac myosins, with 69% amino acid identity. Moreover, these three related myosin genes encode microRNAs (miRNAs) in one of their introns (MYH-␣, miR-208a; MYH-␤, miR-208b; and MYH7b, miR-499) (40, 41). In particular, miR-208a has been shown to be a key mediator of the stress response and a regulator of hypertrophy and electrical conduction in the mouse heart (6, 41). Since MYH7b mRNA levels appear much lower than those of other muscle myosins, the role of MYH7b protein in vertebrate muscle remains unclear.Here, we show that MYH7b expression is controlled transcriptionally by different stimuli, as well as posttranscriptionally, through a unique alternative splicing event. Moreover, we show that miR-499 acts in a regulatory feedback circuit to control MYH7b activity. Our data reveal the complexity of MYH7b gene regulation and unveil a novel mechanism that uncouples host gene-microRNA coexpression. MATERIALS AND METHODSAnimal use and care. C57/BL6 mice used in the experiments were allowed access to standard soy-based rodent chow and water ad libitum. Unless otherwise noted, C57/BL6 male mice 12 weeks of age were used in the experiments. For altered thyroid hormone conditions, mice were fed an iodine-deficient chow supplemented with 0.15% propylthiouracil ([PTU] Harlan Teklad diet composition TD.95125) ad libitum for 30 days. Mice treated with PTU and triiodothyronine (T 3 ) were injected intraperitoneally with 0.2 g/g T 3 on days 29 and 30 of PTU diet ingestion. Four mice from each thyroid condition were sacrificed at 12 weeks of age, and tissues were rapidly excised and froze...

Prdm1a and miR-499 act sequentially to restrict Sox6 activity to the fast-twitch muscle lineage in the zebrafish embryo

“…This begs the question as to how the lineage-restricted expression of miR-499 itself is established. Previous studies in mammalian systems have shown that Mir499 transcription can be repressed by Sox6 (van Rooij et al, 2009;Bell et al, 2010), which would be consistent with slow-lineage-specific mir499 transcription being promoted by Prdm1a activity in zebrafish. To test this possibility, we analysed expression of the mir499 host gene myh7b in wild-type and prdm1a mutant embryos.…”

“…Previous studies have implicated Mir499, a microRNA gene residing in the nineteenth intron of Myh7b, in regulating Sox6 transcript levels in mammalian myofibres, but not in direct repression of Sox6 translation (McCarthy et al, 2009;van Rooij et al, 2009;Bell et al, 2010). MicroRNAs are known to regulate both mRNA stability and translation by targeting sequences in the 3ЈUTRs of mRNAs (Huntzinger and Izaurralde, 2011).…”

“…Consistent with this, overexpression of Sox6 has been shown to repress transcription of the slow-twitch-specific -MHC and troponin genes and to cause a concomitant increase in slow-twitch and decrease in fast-twitch muscle fibres in transgenic mice (van Rooij et al, 2009), and very recent studies have demonstrated that Sox6 binds to conserved cisregulatory elements in slow-twitch fibre genes to represses their transcription in adult fast-twitch muscle (Quiat et al, 2011). Little is known about how the lineage-specific activity of Sox6 is regulated in mammals; however, a role for the microRNA miR-499 has been suggested, based upon the finding that the 3ЈUTRs of human, mouse and rat Sox6 contain between 1 and 4 consensus recognition sites for the miR-499 SEED sequence and the demonstration that miR-499 reduces Sox6 mRNA levels both in vitro and in vivo (McCarthy et al, 2009;van Rooij et al, 2009;Bell et al, 2010).…”

Classification of various muscular tissues using miRNA profiling

“…Because Myh7 and Myh7b are slow-twitch myosin fibers, these miRNAs were detected in slow-twitch muscles. Sex determining region Y-box 6 (Sox6) gene, which represses slow muscle fiber-enriched genes transcription, is a target of miR-208b and miR-499 (5,19,30). Similarly, by analysis of multiple bioinformatics algorithms, a highly conserved binding site for miR-206 is predicted in the 3'UTR of the Sox6 transcripts.…”

Mechanisms of muscle gene regulation in the electric organ ofSternopygus macrurus

“…Emphasis would be placed on studying miRNAs with known functional roles in muscle phenotype regulation. myomiRs, a collection of miRNAs specifically expressed in muscle, have been shown to play roles in myogenesis, muscle fiber size, sarcomere organization and muscle gene expression (Ai et al, 2012;Bell et al, 2010; Callis et al, 2009;Liu et al, 2008;Mishima et al, 2009;Sokol and Ambros, 2005;van Rooij et al, 2007;van Rooij et al, 2009;Zhao et al, 2007). Electrocytes may contain a composition of myomiRs that is different from that in skeletal muscle.…”