Pistoi S, Roland J, Babinet C, Morello D
Department of Immunology, Unité de Biologie du Développement, Institut Pasteur, Paris, France.
Mol Cell Biol. 1996 Sep;16(9):5107-16. doi: 10.1128/MCB.16.9.5107.
We have previously shown that the steady-state level of c-myc mRNA in vivo is primarily controlled by posttranscriptional regulatory mechanisms. To identify the sequences involved in this process, we constructed a series of H-2/myc transgenic lines in which various regions of the human c-MYC gene were placed under the control of the quasi-ubiquitous H-2K class I regulatory sequences. We demonstrated that the presence of one of the two coding exons, exon 2 or exon 3, is sufficient to confer a level of expression of transgene mRNA similar to that of endogenous c-myc in various adult tissues as well as after partial hepatectomy or after protein synthesis inhibition. We now focus on the molecular mechanisms involved in modulation of expression of mRNAs containing c-myc exon 2 sequences, with special emphasis on the coupling between translation and c-myc mRNA turnover. We have undertaken an analysis of expression, both at the mRNA level and at the protein level, of new transgenic constructs in which the translation is impaired either by disruption of the initiation codon or by addition of stop codons upstream of exon 2. Our results show that the translation of c-myc exon 2 is not required for regulated expression of the transgene in the different situations analyzed, and therefore they indicate that the mRNA destabilizing function of exon 2 is independent of translation by ribosomes. Our investigations also reveal that, in the thymus, some H-2/myc transgenes express high levels of mRNA but low levels of protein. Besides the fact that these results suggest the existence of tissue-specific mechanisms that control c-myc translatability in vivo, they also bring another indication of the uncoupling of c-myc mRNA translation and degradation.
我们之前已经表明,体内c-myc mRNA的稳态水平主要受转录后调控机制控制。为了确定参与这一过程的序列,我们构建了一系列H-2/myc转基因系,其中人类c-MYC基因的各个区域置于准普遍存在的H-2K I类调控序列的控制之下。我们证明,两个编码外显子之一,即外显子2或外显子3的存在,足以在各种成年组织中以及在部分肝切除术后或蛋白质合成抑制后赋予转基因mRNA与内源性c-myc相似的表达水平。我们现在专注于参与含有c-myc外显子2序列的mRNA表达调控的分子机制,特别强调翻译与c-myc mRNA周转之间的耦合。我们对新的转基因构建体进行了mRNA水平和蛋白质水平的表达分析,其中翻译通过起始密码子的破坏或在外显子2上游添加终止密码子而受损。我们的结果表明,在分析的不同情况下,c-myc外显子2的翻译对于转基因的调控表达不是必需的,因此它们表明外显子2的mRNA去稳定功能独立于核糖体翻译。我们的研究还揭示,在胸腺中,一些H-2/myc转基因表达高水平的mRNA但低水平的蛋白质。除了这些结果表明存在体内控制c-myc可翻译性的组织特异性机制这一事实外,它们还带来了c-myc mRNA翻译与降解解偶联的另一个迹象。