Abstract
Murine C2C12 myoblasts induced to differentiate into multinucleated myotubes decrease their levels of c-myc mRNA 3-10-fold through posttranscriptional mechanisms that recognize regulatory elements contained in protein-coding sequences in exons 2 and 3 of the mRNA. To determine the mechanism by which these elements mediate c-myc mRNA down-regulation, we examined the regulation of mutant MYC and human beta-globin-MYC fusion mRNAs. Regulation of mRNAs containing MYC exon 2 or 3 is abolished by insertion of an upstream termination codon indicating that regulatory function depends on their translation. Exploiting this translation dependence, we show that pharmacologic inhibition of translation with cycloheximide abolishes the down-regulation of regulated MYC and globin-MYC mRNAs and induces their levels in differentiating C2C12 cells. We exclude the possibility that this induction in mRNA levels results from cycloheximide effects on transcription or processing of parts of the RNA other than the regulatory elements, leading to the conclusion that cycloheximide induction results from mRNA stabilization. We show that the magnitude of cycloheximide induction can be used to estimate turnover rates of mRNAs whose decay is translation-dependent. By using cycloheximide inducibility to examine turnover rates of MYC and globin-MYC mRNAs, we show that the MYC exon 2 and exon 3 regulatory elements, but not MYC 3'-untranslated region or chloramphenicol acetyltransferase coding sequences, mediate accelerated mRNA decay in differentiating, but not undifferentiated, C2C12 cells. We show that these regulatory elements must be translated to confer accelerated mRNA decay and that increased turnover occurs in the cytoplasm and not in the nucleus. Finally, using cycloheximide induction to examine mRNA half-lives, we show that mRNA turnover is increased sufficiently by mechanisms targeting the exon 2 and 3 regulatory elements to account for the magnitude of c-myc mRNA down-regulation during differentiation. We conclude from these results that c-myc mRNA down-regulation during myogenic differentiation is due to translation-dependent mechanisms that target mRNAs containing myc exon 2 and 3 regulatory elements for accelerated decay.
Highlights
Regulated RNA stability plays a critical role in controlling expression of c-myc, a proto-oncogene encoding a transcription factor important in regulating cell proliferation and differentiation
To determine whether translation of the myc exon 3 regulatory element is required for down-regulation, C2C12 cells were stably transfected with MYC(T/N), a MYC cDNA construct from which most 5Ј- and 3Ј-untranslated regions (UTRs) sequences were deleted, or with MYC(Ex2Term3), a construct identical to MYC(T/N) except for the insertion of an in-frame nonsense mutation at codon 262 that prevents translation of about 95% of exon 3 coding sequences, including the exon 3 regulatory element
Northern analysis demonstrated that MYC(T/N) mRNA was maximally down-regulated 3.7-fold during C2C12 differentiation, comparable to the 4.1-fold downregulation in endogenous murine c-myc mRNA. (In figures showing Northern blots from cells transfected with mutant human MYC genes, transgene mRNA is labeled as Hu-MYC to distinguish it from the endogenous murine c-myc mRNA, labeled Mo-myc.) In contrast, MYC(Ex2Term3) mRNA was not down-regulated and even increased over 3-fold, whereas levels of endogenous c-myc mRNA in the same cells were down-regulated 6.7-fold (Fig. 2B)
Summary
Regulated RNA stability plays a critical role in controlling expression of c-myc, a proto-oncogene encoding a transcription factor important in regulating cell proliferation and differentiation. If the regulatory elements mediate down-regulation by accelerating mRNA decay in differentiating C2C12 cells, inhibiting their function should result in stabilization and increased levels of their mRNAs. Since their function is translation-dependent, we tested whether cycloheximide might produce these effects in differentiating C2C12 cells stably transfected with Gm(40 – 262)SVpA or Gm263SVpA, in which the regulatory element from MYC exon 2 or exon 3 was fused to globin, respectively.
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