Evidence That Translation Reinitiation Leads to a Partially Functional Menkes Protein Containing Two Copper-Binding Sites

Abstract

Menkes disease (MD) is an X-linked recessive disorder of copper metabolism. It is caused by mutations in the ATP7A gene encoding a copper-translocating P-type ATPase, which contains six N-terminal copper-binding sites (CBS1-CBS6). Most patients die in early childhood. We investigated the functional effect of a large frameshift deletion in ATP7A (including exons 3 and 4) identified in a patient with MD with unexpectedly mild symptoms and long survival. The mutated transcript, ATP7A(Delta ex3+ex4), contains a premature termination codon after 46 codons. Although such transcripts are generally degraded by nonsense-mediated mRNA decay (NMD), it was established by real-time PCR quantification that the ATP7A(Delta ex3+ex4) transcript was protected from degradation. A combination of in vitro translation, recombinant expression, and immunocytochemical analysis provided evidence that the ATP7A(Delta ex3+ex4) transcript was protected from degradation because of reinitiation of protein translation. Our findings suggest that reinitiation takes place at two downstream internal codons. The putative N-terminally truncated proteins contain only CBS5 and CBS6. Cellular localization and copper-dependent trafficking of the major part of endogenous and recombinant ATP7A(Delta ex3+ex4) proteins were similar to the wild-type ATP7A protein. Furthermore, the ATP7A(Delta ex3+ex4) cDNA was able to rescue a yeast strain lacking the homologous gene, CCC2. In summary, we propose that reinitiation of the NMD-resistant ATP7A(Delta ex3+ex4) transcript leads to the synthesis of N-terminally truncated and at-least-partially functional Menkes proteins missing CBS1-CBS4. This finding--that a mutation that would have been assumed to be null is not--highlights the need to examine the biochemical phenotype of patients to deduce the efficacy of copper therapy.

Biochemical basis of regulation of human copper-transporting ATPases

“…In fact, the truncated ATP7A appeared more sensitive to intracellular copper levels. Compared to the full-length ATP7A, the retention of the truncated ATP7A in Golgi compartment required treatment of cells with higher concentrations of copper chelator (59). This observation could be similar to findings in ATP7B, where the deletion of MBD1,4 appears to facilitate the delivery of copper to the membrane portion of the transporter (41).…”

“…An interesting disease-causing mutation in ATP7A has been described recently by Paulsen and colleagues (59). These authors investigated a large frame-shift deletion in ATP7A identified in a Menkes disease patient with unusually mild symptoms and long survival.…”

Mechanism of escape from nonsense-mediated mRNA decay of human β-globin transcripts with nonsense mutations in the first exon

“…In b-globin, the proximity of the termination event to the AUG and to the 39 mRNP including the poly(A) binding protein C1 within the mRNA ''closed loop'' formation (Wells et al 1998) has been interpreted to mimic a proper position of termination, thus bypassing NMD (Eberle et al 2008;Ivanov et al 2008;Silva et al 2008;Singh et al 2008). In other cases, reinitiation of translation has been shown to play an important role (Zhang and Maquat 1997;Perrin-Vidoz et al 2002;Denecke et al 2004;Buisson et al 2006;Paulsen et al 2006).…”

Ohtahara syndrome in a family with an ARX protein truncation mutation (c.81C>G/p.Y27X)

“…Translation re-initiation may explain the relatively 'mild' non-malformation phenotype observed in this family, consistent with the observations made in other diseases. 12 However, this explanation is subject to the proposition that the in vivo c.81C4G containing ARX mRNA escapes degradation by the nonsense-mediated decay (NMD) pathway. We predict that NMD partially degrades the c.81C4G ARX mRNA, as is seen with other PTC containing mRNAs, 13 and is most likely protected from complete degradation because of the proximity of the PTC to the translation initiation codon.…”