Abnormal muscle development in the heldup3 mutant of Drosophila melanogaster is caused by a splicing defect affecting selected troponin I isoforms.

Abstract

The troponin I (TnI) gene of Drosophila melanogaster encodes a family of 10 isoforms resulting from the differential splicing of 13 exons. Four of these exons (6al, 6a2, 6bl, and 6b2) are mutually exclusive and very similar in sequence. TnI isoforms show qualitative specificity whereby each muscle expresses a selected repertoire of them. In addition, TnI isoforms show quantitative specificity whereby each muscle expresses characteristic amounts of each isoform. In the mutant heldup3, the development of the thoracic muscles DLM, DVM, and TDT is aborted. The mutation consists of a one-nucleotide displacement of the 3' AG splice site at the intron preceding exon 6bl, resulting in the failure to produce all exon 6bl-containing TnI isoforms. These molecular changes in a constituent of the thin filaments cause the selective failure to develop the DLM, DVM, and TDT muscles while having no visible effect on other muscles wherein exon 6b1 expression is minor.We have reported that the haplolethal (HL) region of the Shaker gene complex (ShC) of Drosophila melanogaster (13) harbors the structural gene for troponin I (TnI) (2) and ascribed the heldup (hdp) mutations, originally described by Deak (10), to the TnI gene on the basis of genetic complementation tests. Later, Beall and Fyrberg (4) showed that some hdp mutants lack several TnI isoforms. However, since the mutant phenotype consists in the severe depletion of selected muscles, it is not clear whether the lack of specific TnI isoforms is the cause or the consequence of the muscle defect.We find that the allele mutation hdp3 consists of a single nucleotide change at the A(G/G) 3' border of the intron preceding exon 6bl of TnI, which is converted to A(A/G). The splicing complex in the mutant does not seem to recognize the new splice site and consequently does not express exon 6bl-containing RNA isoforms in any muscle, including those that do not show the mutant phenotype. The muscles not affected by the absence of exon 6bl are those in which the expression of this exon would have been quantitatively minor.TpI is a constituent of the thin filaments and plays a regulatory role in muscle contraction (33). It binds to actin, blocking the actin-myosin interaction in the resting state.Upon Ca2' entry, TnI shifts to interact with troponin C, leaving actin free and allowing the relative displacement of thin and thick filaments. Muscle physiology in insects shows peculiar features which sustain their extraordinary performance during wing beat (25). In the insect fibrillar flight muscles (DLM and DVM), calcium alone is not enough to produce full activation. The Ca2+-activated muscles are further activated in both tension and ATPase activity by small strains (1 to 3%) of their fibers. This property, first shown in the giant waterbug Lethocerus sp. (32), is also seen in D. melanogaster (23) and enables the wings to beat at high frequency. It is expected that the diversity of mechanical * Corresponding author. t Present address:

Functional recovery of troponin I in a Drosophila heldup mutant after a second site mutation.

“…Four suppressors mapped to chromosome II in the interval between the markers rd-pr, one to chromosome III between the markers red-sbd, and one to chromosome X between C E' *4 y .T . Set j rt s jE}ssvan-s -Za (Barbas et al, 1993), indicating that the suppression by D3 is allele specific. At the light microscope level (Figure 1), the hdp2 mutation ( Figure 1B) causes a major collapse of IFM leaving the tubular muscles in the body with minor or unnoticeable defects that, in the case of the larval and adult tubular muscles, still allow proper function.…”

“…TDT and IFM muscles from Canton-S, hdp2, and hdp2 D3 genotypes were dissected and analyzed separately. To identify the full repertoire of TnI isoforms, we used the J2 antibody described in Barbas et al (1993). Figure 4 shows that the exon 3-containing group of isoforms as well as the non-exon 3-containing group were present in the three genotypes in similar amounts.…”

“…According to these type of data, the carboxy terminus of Tnl sustains the interactions with TnC and actin as well as the Ca++-dependent ATPase regulation whereas the amino terminus is needed to maintain the stability of the ternary complex TnI-TnC-TnT and has no ATPase regulation. However, TnI is present in a variety of forms encoded by three different genes in the mouse or 10 splice variants from a single gene in Drosophila (Barbas et al, 1993). At isoforms may be present in the same muscle.…”

“…Actin, a-actinin, tropomyosin, TnT, TnI, and TnC have all been identified and sequenced (see Bernstein et al, 1993 for a review). In addition, insect muscles contain peculiar myofibrillar proteins such as arthrin and troponin H. In view of the large diversity of TnI isoforms and their muscle-specific expression pattern (Barbas et al, 1993) it seems plausible that this protein(s) plays distinct functional roles that the in vitro studies carried out so far cannot discriminate.…”

Transcription ofDrosophilaTroponin I Gene Is Regulated by Two Conserved, Functionally Identical, Synergistic Elements

“…Monospecific anti-Troponin I (Barbas et al, 1993) and anti-Ariadne (Aguilera et al, 2000) antibodies were used at 1:1000 and 1:75 dilutions respectively. Signal was developed by the chemiluminiscent ECL method (Amersham Biosciences).…”