Aberrant Splicing of an Alternative Exon in the Drosophila Troponin-T Gene Affects Flight Muscle Development

Article excerpt


During myofibrillogenesis, many muscle structural proteins assemble to form the highly ordered contractile sarcomere. Mutations in these proteins can lead to dysfunctional muscle and various myopathies. We have analyzed the Drosophila melanogaster troponin T (TnT) up^sup 1^ mutant that specifically affects the indirect flight muscles (IFM) to explore troponin function during myofibrillogenesis. The up^sup 1^ muscles lack normal sarcomeres and contain "zebra bodies," a phenotypic feature of human nemaline myopathies. We show that the up^sup 1^ mutation causes defective splicing of a newly identified alternative TnTexon (10a) that encodes part of the TnT C terminus. This exon is used to generate a TnT isoform specific to the IFM and jump muscles, which during IFM development replaces the exon 10b isoform. Functional differences between the 10a and 10b TnT isoforms may be due to different potential phosphorylation sites, none of which correspond to known phosphorylation sites in human cardiac TnT. The absence of TnT mRNA in up^sup 1^ IFM reduces mRNA levels of an IFM-specific troponin I (TnI) isoform, but not actin, tropomyosin, or troponin C, suggesting a mechanism controlling expression of TnT and TnI genes may exist that must be examined in the context of human myopathies caused by mutations of these thin filament proteins.

TROPONIN T (TnT), troponin I (TnI), and troponin C (TnC) form the troponin complex, which with tropomyosin (Tm) act as a regulatory switch for striated muscle contraction (reviewed in Gordon et al. 2000). Striated muscle contraction, including that of Drosophila indirect flight muscles (IFM) and the tergal depressor of the trochanter (TDT) or jump muscle (Peckham et al. 1990), is activated by Ca21, although the IFM also require an applied strain. At low intracellular Ca21 concentrations, the TnI subunit inhibits actomyosin activity. When intracellular Ca21 concentration is increased, Ca21 ions bind to TnC, leading to conformational changes in the Tn-Tm complex, removing the TnI inhibition, and activating actomyosin crossbridge activity. Biochemical studies have shown that TnT is required for both complete inhibition and activation of actomyosin activity in the absence or presence of Ca21 (Potter et al. 1995; Oliveira et al. 2000).

The importance of TnT is underlined by the discovery of mutations that cause human familial cardio- and nemaline myopathies (Perry 1998; Johnston et al. 2000; Roberts and Sigwart 2001; Morimoto et al. 2002; Towbin and Bowles 2002).

Mutations of TnT genes in other model genetic organisms can result in severe muscle phenotypes and may also affect the expression of other thin filament protein genes. In Caenorhabditis elegans TnT (CeTnT-1), gene mutations can cause detachment of body wall muscles. It has been argued that this results from prolonged force development caused by an inability of the muscles to relax once Ca21-dependent muscular activation has occurred (Myers et al. 1996; Mcardle et al. 1998). Studies of silent heart mutants in the zebrafish TnT2 gene indicate that the muscle phenotype arises by reduced expression and accumulation of TnT2 message and protein combined with reductions of other thin filament proteins (Sehnert et al. 2002). Recent studies of D. melanogaster TnI mutations, heldup-2, hdp^sup 2^ (where muscle detachment occurs once muscle contraction is activated), and hdp^sup 3^ (an IFM-TDT-specific TnI null), suggest that hypercontracted muscle phenotypes can be associated with coordinated reductions in message and protein levels of other thin filament proteins (Nongthomba et al. 2003, 2004). To understand how TnTmutants cause defective muscle development requires a more complete understanding of how they can affect the expression and assembly of other muscle proteins.

The IFM are dispensable for survival under laboratory conditions and there are IFM-specific isoforms of many muscle structural proteins, making Drosophila IFM an effective genetic model for muscle studies (Vigoreaux 2001; Nongthomba et al. …