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J. Biol. Chem., Vol. 280, Issue 14, 13241-13249, April 8, 2005

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Cellular Fate of Truncated Slow Skeletal Muscle Troponin T Produced by Glu¹⁸⁰ Nonsense Mutation in Amish Nemaline Myopathy^*

Xin Wang, Qi-Quan Huang, Mark T. Breckenridge, Aihua Chen, Thomas O. Crawford¶, D. Holmes Morton||, and Jian-Ping Jin**

From the Section of Molecular Cardiology, Evanston Northwestern Healthcare and Northwestern University Feinberg School of Medicine, Evanston, Illinois 60201, the Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, the ^¶Departments of Neurology and Pediatrics, Johns Hopkins University, Baltimore, Maryland 21287, and the ^||Clinic for Special Children, Strasburg, Pennsylvania 17579

A nonsense mutation at codon Glu¹⁸⁰ in exon 11 of slow skeletal muscle troponin T (TnT) gene (TNNT1) causes an autosomal-recessive inherited nemaline myopathy. We previously reported the absence of intact or prematurely terminated slow TnT polypeptide in Amish nemaline myopathy (ANM) patient muscle. The present study further investigates the expression and fate of mutant slow TnT in muscle cells. Intact slow TnT mRNA was readily detected in patient muscle, indicating unaffected transcription and RNA splicing. Sequence of the cloned cDNAs revealed the single nucleotide mutation in two alternatively spliced isoforms of slow TnT mRNA. Mutant TNNT1 cDNA is translationally active in Escherichia coli and non-muscle eukaryotic cells, producing the expected truncated slow TnT protein. The mutant mRNA was expressed at significant levels in differentiated C₂C₁₂ myotubes, but unlike intact exogenous TnT, truncated slow TnT protein was not detected. Transfective expression in undifferentiated myoblasts produced slow TnT mRNA but not a detectable amount of truncated or intact slow TnT proteins, indicating a muscle cell-specific proteolysis of TnT when it is not integrated into myofilaments. The slow TnT-(1–179) fragment has substantially lower affinity for binding to tropomyosin, in keeping with the loss of one of two tropomyosin-binding sites. Our findings suggest that inefficient incorporation into myofilament is responsible for the instability of mutant slow TnT in ANM muscle. Rapid degradation of the truncated slow TnT protein, rather than instability of the nonsense mRNA, provides the protective mechanism against the potential dominant negative effect of the mutant TnT fragment.

Received for publication, December 6, 2004 , and in revised form, January 10, 2005.

^* This work was supported in part by National Institutes of Health NIAMS Grant AR-048816 and NICHD Grant HD-044824 (to J.-P. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY762903 and AY762904.

^** To whom correspondence should be addressed. Tel.: 847-570-1960; Fax: 847-570-1865; E-mail: jpjin{at}northwestern.edu.

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