Yeilding N M, Procopio W N, Rehman M T, Lee W M
Department of Medicine and the Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 1998 Jun 19;273(25):15749-57. doi: 10.1074/jbc.273.25.15749.
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.
诱导分化为多核肌管的小鼠C2C12成肌细胞通过识别mRNA外显子2和3中蛋白质编码序列所含调控元件的转录后机制，使其c-myc mRNA水平降低3至10倍。为了确定这些元件介导c-myc mRNA下调的机制，我们研究了突变型MYC和人β-珠蛋白-MYC融合mRNA的调控。含有MYC外显子2或3的mRNA的调控因上游终止密码子的插入而被消除，这表明调控功能取决于它们的翻译。利用这种对翻译的依赖性，我们发现用环己酰亚胺进行翻译的药理抑制消除了受调控的MYC和珠蛋白-MYC mRNA的下调，并在分化的C2C12细胞中诱导了它们的水平。我们排除了mRNA水平的这种诱导是由环己酰亚胺对除调控元件之外的RNA其他部分的转录或加工的影响所导致的可能性，从而得出结论，环己酰亚胺诱导是由mRNA稳定化引起的。我们表明，环己酰亚胺诱导的幅度可用于估计其衰变依赖于翻译的mRNA的周转率。通过使用环己酰亚胺诱导性来检测MYC和珠蛋白-MYC mRNA的周转率，我们发现MYC外显子2和外显子3调控元件，而非MYC 3'-非翻译区或氯霉素乙酰转移酶编码序列，在分化而非未分化的C2C12细胞中介导mRNA的加速衰变。我们表明，这些调控元件必须被翻译才能赋予加速的mRNA衰变，并且增加的周转率发生在细胞质而非细胞核中。最后，利用环己酰亚胺诱导来检测mRNA半衰期，我们发现通过靶向外显子2和3调控元件的机制，mRNA周转率充分增加，足以解释分化过程中c-myc mRNA下调的幅度。我们从这些结果得出结论，肌源性分化过程中c-myc mRNA的下调是由于依赖于翻译的机制，该机制靶向含有myc外显子2和3调控元件的mRNA以加速衰变。
