Biochemical and Biophysical Research Communications, Vol.502, No.3, 422-428, 2018
Readthrough of ACTN3 577X nonsense mutation produces full-length alpha-actinin-3 protein
The ACTN3 gene encodes alpha-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 alpha-actinin-3 protein production due to targeted degradation of the mutant transcript by the cellular nonsensemediated mRNA decay (NMD) system, which requires mRNA splicing. In humans, alpha-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 fulllength alpha-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 X-pre, 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 X-pre gene, expressed exogenous truncated alpha-actinin-3 protein, indicating NMD-mediated suppression of exogenous X-pre expression. Cells treated with aminoglycosides produced exogenous full-length alpha-actinin-3 protein in X-transfected cells, but not in X-pre-transfected cells. The NMD inhibitor caffeine prevented suppression of X-pre expression and thereby induced production of full-length alpha-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. (C) 2018 Elsevier Inc. All rights reserved.