Differential regulation of multiple alternatively spliced transcripts of MyoD.

Splice variants of the basic helix-loop-helix myoblast determination factor (myoD) have not been previously found in vertebrates. Here we report the identification and characterization of three alternative transcripts of a myoD paralogue from the tiger pufferfish (Takifugu rubripes). The T. rubripes myoD1 gene (TmyoD1) has 3 exons and 2 introns and it is present on scaffold 104, in a region of conserved synteny with zebrafish. The isoform TMyoD1-alpha is a putative protein of 281 residues that contains the basic, helix-loop-helix and helix III domains and shares 61%, 56%, 51%, 49% and 56% overall identity with zebrafish, Xenopus, mouse, human and chicken MyoD1, respectively. TMyoD1-beta arises from an alternative 3' splice site and differs from TMyoD1-alpha by a 26-residue insertion adjacent to helix III, which is one of the functional domains required for chromatin remodelling. The third alternative transcript, TmyoD1-gamma, retains intron I and has two premature termination codons far from the 3'-most exon-exon junction. TmyoD1-gamma is therefore likely to be degraded by nonsense-mediated decay, an important widespread post-transcriptional mechanism that regulates transcript levels. Analysis of gene expression by qPCR revealed that TmyoD1-alpha was the most abundant transcript in fast and slow myotomal muscle. TmyoD1-alpha expression was 2-fold higher in fast muscle of juvenile fish that were actively producing new myotubes compared to adult stages that had stopped recruiting fast muscle fibres. A similar expression pattern was observed for TmyoD1-alpha in slow muscle but the differences were not significant. Transcript levels of TmyoD1-gamma only varied significantly in fast muscle and were 5-fold higher in adult compared to juvenile stages. Significant differences in expression of TmyoD1 splice variants were also observed during embryonic development. The differential expression of three alternative transcripts of myoD1 in developing and adult myotomal muscle of T. rubripes supports the hypothesis that diversity generated by alternative splicing may be of functional significance in muscle development in this species.