Abstract
BackgroundThree different gene clusters code for the muscle-specific miRNAs miR-206, miR-1 and miR-133a/b. The two miR-1/133a clusters generate identical mature miR-1 and miR-133a miRNAs in heart and skeletal muscle, while the cognate miR-206/133b cluster is exclusively expressed in skeletal muscle. Since sequences of the miRNAs miR-133a and miR-133b are almost identical, it seems likely that they share potential targets. Similarly, miR-1 and miR-206 are structurally related and contain identical seed sequences important for miRNA-target recognition. In the past, different functions of these miRNAs were suggested for development, function and regeneration of skeletal muscle using different in vivo and in vitro models; however, mutants lacking the complete miR-206/133b cluster, which generates a single pri-miRNA constituting a functional unit, have not been analyzed.MethodsWe generated miR-206/133b knock-out mice and analyzed these mice morphologically; at the transcriptome and proteome level to elucidate the contribution of this miRNA cluster for skeletal muscle development, differentiation, regeneration in vivo; and by systematic analysis. In addition, we studied the consequences of a genetic loss of miR-206/133b for expression of Pax7 and satellite cell differentiation in vitro.ResultsDeletion of the miR-206/133b cluster did not reveal any obvious essential function of the miRNA-cluster for development and differentiation of skeletal muscle. Careful examination of skeletal muscles of miR-206/133b mutants revealed no structural alterations or molecular changes at the transcriptome and proteome level. In contrast to previous studies, deletion of the miR-206/133b cluster did not impair regeneration of skeletal muscle in mdx mice. Likewise, differentiation of miR-206/133b deficient satellite cells in vitro was unaffected and no change in Pax7 protein concentration was apparent.ConclusionsWe conclude that the miR-206/133b cluster is dispensable for development, function and regeneration of skeletal muscle, probably due to overlapping functions of the related miR-1/133a clusters, which are strongly expressed in skeletal muscle. We reason that the miR-206/133b cluster alone is not an essential regulator of skeletal muscle regeneration, although more subtle functions might exist that are not apparent under laboratory conditions.
Highlights
Three different gene clusters code for the muscle-specific miRNAs miR-206, miR-1 and miR-133a/b
The mouse genome contains two miR-1/ 133a gene clusters located on chromosome 2 and 18, giving rise to identical mature miR-1 and miR-133a miRNAs while the structurally related miR-206/133b cluster is located on mouse chromosome 1
Deletion of both miR-1/133a clusters, which are expressed in heart and all skeletal muscles, results in early embryonic lethality due to defects in heart development caused by the failure to restrict myocardin expression and concomitant upregulation of smooth muscle genes [2]
Summary
Three different gene clusters code for the muscle-specific miRNAs miR-206, miR-1 and miR-133a/b. MiR-1 and miR-206 do not differ in the seed sequence that is assumed to determine target specificity of miRNAs [1] Deletion of both miR-1/133a clusters, which are expressed in heart and all skeletal muscles, results in early embryonic lethality due to defects in heart development caused by the failure to restrict myocardin expression and concomitant upregulation of smooth muscle genes [2]. Deletion of both miR-1 copies leads to cardiac defects with partially penetrant neonatal lethality [3] but did not cause a major skeletal muscle phenotype, which was attributed to the remaining expression of miR-206 in skeletal muscle. Deletion of both miR-133a copies affects cardiomyocyte proliferation and heart physiology [4] and results in a centronuclear skeletal myopathy and a shift of muscle fiber identity from glycolytic to oxidative muscle fibers in the soleus muscle [5]
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