Coding Elements in Exons 2 and 3 Target c-myc mRNA Downregulation during Myogenic Differentiation

Abstract

Downregulation in expression of the c-myc proto-oncogene is an early molecular event in differentiation of murine C2C12 myoblasts into multinucleated myotubes. During differentiation, levels of c-myc mRNA decrease 3- to 10-fold despite a lack of change in its transcription rate. To identify cis-acting elements that target c-myc mRNA for downregulation during myogenesis, we stably transfected C2C12 cells with mutant myc genes or chimeric genes in which various myc sequences were fused to the human β-globin gene or to the bacterial chloramphenicol acetyltransferase (CAT) gene. Deletion of coding sequences from myc exon 2 or exon 3 abolished downregulation of myc mRNA during myogenic differentiation, while deletion of introns or sequences in the 5′ or 3′ untranslated regions (UTRs) did not, demonstrating that coding elements in both exons 2 and 3 are necessary for myc mRNA downregulation. Fusion of coding sequences from either myc exon 2 or 3 to β-globin mRNA conferred downregulation onto the chimeric mRNA, while fusion of myc 3′ UTR sequences or coding sequences from CAT or ribosomal protein L32 did not, demonstrating that coding elements in myc exons 2 and 3 specifically confer downregulation. These results present the apparent paradox that coding elements in either myc exon 2 or myc exon 3 are sufficient to confer downregulation onto β-globin mRNA, but neither element alone was sufficient for myc mRNA downregulation, suggesting that some feature of β-globin mRNA may potentiate the regulatory properties of myc exons 2 and 3. A similar regulatory function is not shared by all mRNAs because fusion of either myc exon 2 or myc exon 3 to CAT mRNA did not confer downregulation onto the chimeric mRNA, but fusion of the two elements together did. We conclude from these results that two myc regulatory elements, one exon 2 and one in exon 3, are required for myc mRNA downregulation. Finally, using a highly sensitive and specific PCR-based assay for comparing mRNA levels, we demonstrated that the downregulation mediated by myc exons 2 and 3 results in a decrease in cytoplasmic mRNA levels, but not nuclear mRNA levels, indicating that regulation is a postnuclear event.