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,Jian-Ping Jin

doi:10.1074/jbc.m413696200

Abstract

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.

Highlights

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
The Mutant Slow TnT mRNA Is Present at Significant Amounts in Amish nemaline myopathy (ANM) Patient Muscle—Because the limited quantity of diagnostic biopsy muscle samples precludes the use of Northern blot to detect and quantify the amount of slow TnT mRNA, we applied reverse transcription (RT)-PCR to examine the mRNA with the opportunity to clone cDNA from the mutant mRNA
The results verified the authenticity of the cloned cDNA and demonstrate that the CT3 epitope is preserved in the truncated slow TnT, confirming that the negative CT3 Western blots of ANM patient muscle biopsy samples indicates the absence of truncated slow TnT [13]

Summary

The abbreviations used are

TnC, troponin C; TnI, troponin I; TnT, troponin T; ANM, Amish nemaline myopathy; CMV, cytomegalovirus; FBS, fetal bovine serum; mAb, monoclonal antibody; MHC, myosin heavy chain; RT, reverse transcription; DMEM, Dulbecco’s modified Eagle’s medium. These protein binding data [17]. Transfective expression in undifferentiated myoblasts do not produce 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 These results demonstrate instability of slow TnT-(1–179) within muscle cells, suggesting that the loss of TnI, TnC, and one tropomyosin-binding site restricts incorporation of mutant slow TnT into the myofilament, which in turn leads to accelerated degradation. Rapid degradation of TnT-(1–179) protein, rather than loss of the nonsense mRNA, is responsible for the absence of a dominant effect of this mutation

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION