Subfunctionalization of the salmonid myostatin gene family

Abstract

Myostatin is a potent negative regulator of striated muscle growth in mammals, although its function in other vertebrates is not well known and is believed to be far more diverse. Previous phylogenetic analyses have identified four distinct paralogs in salmonid fishes, MSTN-1a, -1b, -2a and --2b, whose functions appear to be actively diverging based on their unique patterns of gene expression and transcript processing. These conclusions, however, are based upon a limited number of salmonid paralogs. We therefore cloned several paralogs from additional salmonid species and characterized the genomic organization and putative splice sites for each. Comparative mapping revealed a conserved organization of exons, introns and coding frames, although paralog-and taxa-specific differences were noted. For instance, MSTN-1 paralogs were similarly organized while MSTN-2a and -2b structures differed considerably. All MSTN-2b genes included in-frame stop codons, confirming pseudogenization across the family, although the underlying indels differed between taxa. The MSTN-2b orthologs are therefore useful candidates for defining phylogenetic relationships within the family. Previous studies identified tissue-specific alternative processing of MSTN-2a, but not MSTN-2b transcripts, in rainbow trout. Multiple sequence alignments of MSTN-2 intron/exon boundaries failed to identify conserved splice sites, indicating that MSTN-2a and -2b lack the basic ciselements necessary for transcript processing. Thus, the limited processing of MSTN-2a transcripts to rainbow trout brains likely occurs in other salmonids as well and may be regulated by neural-specific factors that bind YCAY motifs that direct the spliceosome. In fact, MSTN-2a paralogs contain significantly more of these motifs than do the unspliced MSTN-2b paralogs or other comparably sized genes. These data together suggest that the salmonid myostatin gene family is a potentially novel resource for investigating the functional divergence of duplicated genes, particularly as the mechanisms that regulate gene expression, transcript processing and protein structure are actively subfunctionalizing.