| Summary: | MAFbx is an E3 ubiquitin ligase which plays important roles in myogenesis and muscle atrophy. We characterised the Atlantic salmon MAFbx gene, identifying two alternatively spliced MAFbx isoforms. The mRNA sequence of Atlantic salmon MAFbx-α is 1698 nucleotides long including a 134 bp 5′ UTR and 1065 bp coding sequence which encodes a 355 amino acid protein with a predicted mass of 41,657 Da and pI 8.74. Two different 3′ UTRs were identified of 495 and 314 bp in length. MAFbx-β is produced by the removal of the 116 bp exon 2 from MAFbx-α, resulting in a frame shift mutation and introduction of a premature stop codon. In contrast to mammals, MAFbx-α and β were ubiquitously expressed in all salmon tissues examined. In vivo, expression was 600-fold (MAFbx-α) and 200-fold (MAFbx-β) higher in fasted individuals than following 21 days refeeding to satiation. In primary myogenic cell cultures, MAFbx-α mRNA was highest in differentiated myotubes while MAFbx-β mRNA had peak expression in mono-nucleated cells. Starving cells of serum and amino acids resulted in a 6-fold increase in MAFbx-α, whereas MAFbx-β remained similar to control levels. In starved cells, MAFbx-α mRNA levels declined in response to amino acid, IGF-I and IGF-II treatments whereas MAFbx-β levels only decreased in response to IGF-I. Addition of amino acids and IGF or insulin to starved cells increased MAFbx-β levels after 12 and 24 h. These results indicate that regulation of MAFbx in Atlantic salmon occurs at both the transcriptional and post-transcriptional level through production of the alternatively spliced MAFbx-β, which is a likely target for non-sense mediated decay.
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