The ACTN3 gene encodes α-actinin-3 protein, which stabilizes the contractile apparatus at the Z-line in skeletal muscle cell fast fibers. A nonsense mutation of the arginine (R) at the codon for amino acid 577 of the ACTN3 gene generates a premature termination codon (PTC) and produces the R577X polymorphism in humans (X specifies translational termination). The ACTN3 577X genotype abolishes α-actinin-3 protein production due to targeted degradation of the mutant transcript by the cellular nonsense-mediated mRNA decay (NMD) system, which requires mRNA splicing. In humans, α-actinin-3 deficiency can decrease sprinting and power performance as well as skeletal muscle mass and strength. Here we investigated whether suppression of the in-frame PTC induced by treatment with the aminoglycosides gentamicin and G418 that promote termination codon readthrough could allow production of full-length α-actinin-3 protein from ACTN3 577X. We constructed expression plasmids encoding mature mRNA that lacks introns or pre-mRNA, which carries introns for the ACTN3 577X gene (X and Xpre, respectively) and transfected the constructs into HEK293 cells. Similar constructs for the ACTN3 577R gene were used as controls. HEK293 cells carrying the X gene, but not the Xpre gene, expressed exogenous truncated α-actinin-3 protein, indicating NMD-mediated suppression of exogenous Xpre expression. Cells treated with aminoglycosides produced exogenous full-length α-actinin-3 protein in X-transfected cells, but not in Xpre-transfected cells. The NMD inhibitor caffeine prevented suppression of Xpre expression and thereby induced production of full-length α-actinin-3 protein in the presence of aminoglycoside. Together these results indicate that the ACTN3 R577X polymorphism could be a novel target for readthrough therapy, which may affect athletic and muscle performance in humans.
Keywords: ACTN3; Aminoglycoside; Nonsense-mediated mRNA decay; Premature termination codon; Readthrough; α-Actinin-3.
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